Monitoring health and disease status using clonotype profiles

ABSTRACT

There is a need for improved methods for determining the diagnosis and prognosis of patients with conditions, including autoimmune disease and cancer, especially lymphoid neoplasms, such as lymphomas and leukemias. Provided herein are methods for using DNA sequencing to identify personalized, or patient-specific biomarkers in patients with lymphoid neoplasms, autoimmune disease and other conditions. Identified biomarkers can be used to determine and/or monitor the disease state for a subject with an associated lymphoid disorder or autoimmune disease or other condition. In particular, the invention provides a sensitive method for monitoring lymphoid neoplasms that undergo clonal evolutions without the need to development alternative assays for the evolved or mutated clones serving as patient-specific biomarkers.

This application is a continuation of co-pending U.S. application Ser.No. 13/100,365 filed 4 May 2011 “application '365”), application '365being a continuation-in-part of U.S. application Ser. No. 12/615,263filed 9 Nov. 2009 (now U.S. Pat. No. 8,236,503) which claims priorityfrom U.S. provisional application Ser. No. 61/112,693 filed 7 Nov. 2008,and application '365 claiming priority from U.S. provisional applicationSer. No. 61/332,175 filed 6 May 2010, Ser. No. 61/455,743 filed 25 Oct.2010, and Ser. No. 61/446,822 filed 25 Feb. 2011, all of which areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The invention relates generally to monitoring health and diseaseconditions of an individual by molecular measurements, and moreparticularly, to monitoring health and disease conditions of anindividual by measuring profiles immune system molecules using highthroughput DNA sequencing.

BACKGROUND OF THE INVENTION

The adaptive immune system, comprising humoral (or B cell mediated) andcytotoxic (or T cell-mediated) responses, has evolved to attack specificmolecular features on their respective targets. The occurrence of oneresponse to a specific target provides a host with “memory” of it,giving it a capability to mount a stronger response if the same targetwere to appear another time. Usually any protein or polysaccharide canserve as the target for some subset of the adaptive immune responsecells or their products that recognize specific molecular features, orepitopes, on the target.

Since autoimmune disease involves the recognition by some component ofthe adaptive immune system to self targets, aspects of the adaptiveimmune system have been examined to aid in diagnosis and prognosis ofsuch diseases. Using standard immunological techniques, the humoralimmune system has been investigated by looking for circulatingautoantibodies. Autoantibodies, like antinuclear, anti-dsDNA, andrheumatoid factor, have been identified for several diseases. Theseantibodies may not themselves be pathological, nor is the target theyrecognize in the body necessarily the same as that tested for in vitro;however, measurement of their levels aids in the diagnosis and in somecases has some prognostic and treatment implications.

Another methodology to study the adaptive immune system in autoimmuneand lymphoid diseases is based on the analysis of the diversity of theadaptive immune cells. Activation of the adaptive immune cells leads totheir clonal expansion. Evidence of this clonal expansion is usuallyobtained by amplification from the blood RNA or DNA of part of thenucleic acid sequence coding for the antigen recognition region. Forexample, PCR primers to amplify sequences that have a specific V segmentof the p chain in T-cell receptor (analogous to antibody heavy chain)are used to amplify the J segments or J and D segments connected to thespecific V segment. When a diverse cell population is present it isexpected to amplify fragments with a distribution of slightly differentsize amplicons, but clonal expansion causes specific sizes to becomeenriched and thus more intense as visualized as bands on a gel. In thetechnique called “spectratyping” each of the V segments is amplifiedwith the J and D segments to assess whether any of these amplicons showsa clonal expansion.

One problem of the spectratyping approach is that many distinctsequences can have the same length and hence are indistinguishable.Therefore only dramatic clonal expansion can be discerned byspectratyping. There is need to improve methods of diagnosing and aidingprognosis of autoimmune disease and autoimmune disease states as well asother diseases for which the immune system plays a central role.

While additional specificity in profiling the immune system would be ofgreat utility in allowing its impact on human health to be betterpredicted, still greater utility would be delivered if methods weredeveloped that would allow the specific T and B cells involved indisease processes to be identified even if those particular sequenceshad never before been observed. The vast diversity of the immune systemprovides it with an immense reserve of potentially useful cells but alsopresents a challenge to the researcher trying to use this repertoire forpredictive purposes. Any single sequence targeting an antigen is one ofa vast number that could be involved with and/or correlated to thedisease process in a given individual. Methods that would identify whichof the many cells in a given individual are involved with diseaseprocesses would be of great value to human health.

Immune cells profiling also has utility in the diagnosis and managementof cancers. Treatment of cancers frequently involves the evaluation ofresponse to treatment as well as monitoring for the recurrence ofdisease. Most common methodologies to monitor response and cancerrecurrence are radiographic evaluations as well as blood biomarkers. Forexample, CT scans are frequently used to monitor cancer recurrence inmultiple diseases including colon cancer. Similarly, protein biomarkers,like PSA and CEA, are blood biomarkers used to follow prostate and coloncancers. Specific genomic rearrangements generate another attractivetarget to use for following cancer cells. For example, the BCR-ABLtranslocation present in the vast majority of Chronic MyelogenousLeukemia (CML) patients has emerged as an analyte to assess the state ofthe disease. The specificity of the translocation to the leukemic cellsand its amenability to be assayed by PCR technology allowed for thegeneration of a highly specific and sensitive test that is now usedroutinely to monitor CML patients.

Immune cell (or clonotype) profiling can be used to generate markers forlymphoid neoplasms. Cancer in the lymphoid cell lineage is aheterogeneous set of clinical diseases often reflecting thedevelopmental stage of the cell that have undergone the transformationto a cancerous cell. Acute Lymphoblastic Leukemia (ALL) most oftenarises in immature lymphocytes. On the other hand, Multiple Myeloma (MM)occurs in plasma cells that have differentiated to produce antibodies.Similarly the different types of lymphomas often reflect different celldevelopmental stages. These diseases occur in different age groups, havedifferent prognosis and mortality, and can be treated with distinctregimens.

These diseases are frequently treated with chemotherapy, radiotherapy,and/or bone marrow transplant. The disease recurrence is then monitoredby different methods depending on the particular clinical situation.These methods include the assessment of blood and/or bone marrow usingstandard blood counts and morphology, flow cytometry (FCM) using cellsurface markers, protein electrophoresis, as well as moleculartechniques like PCR and FISH. In addition, radiographic studies like CTand PET scanning are frequently utilized for monitoring the recurrenceof some of the lymphoid cancers. These methods suffer from invasiveness(bone marrow), cost and radiation risk, and/or lack of sensitivity.

Some molecular markers specific to a cancer cell detectable by PCR in asensitive manner are present in a fraction of the lymphoid neoplasms.For example BCR-ABL is present in a fraction of ALL patients and it canserve as a marker to monitor for the relapse of the tumor.Unfortunately, for the majority of patients there are no such markersthat can be used for sensitive and specific detection of relapse. FCMcan be used to detect Minimum Residual Disease (MRD) which is useful forprognostic purposes. In this technique using multi-color Flow ActivatedCell Sorting (FACS), a cancer cell can be identified by the virtue ofthe particular cell surface markers that it has. The sensitivity of thistechnique in the hands of experts is limited to <10⁻⁴, (1 cancer cell in10,000 normal cells) and markers present at one time point may disappearlater. Therefore FCM is generally not useful in detecting early relapsein blood samples.

PCR provides a sensitive methodology for detection of specific sequencesand it has been used to detect the particular rearrangement in B cellreceptor (BCRs) or T cell receptors (TCRs) of the cancer cell. Thistechnique capitalizes on the fact that B or T cell receptors in alymphocyte are created after imperfect recombination events thatgenerate unique sequences for the different lymphocytes. For example, aTCR is comprised of TCRα and TCRβ chains. TCRα is created through therecombination that links one of several different V regions to one ofseveral J regions. Similarly TCRβ is created through recombination thatcreates one V, D, and J segment in tandem. In both cases therecombination is often not perfect and some bases can be deleted fromthe germ line segment sequences and other bases (called the N and Pbases) may be added. The sequence between the V and J segments isreferred to as the NDN region.

These sequences can then serve as a tag for these lymphocytes and theirprogeny. Since these recombination events also occur in the cells thatultimately become malignant, unique sequences of the B and T cellreceptors can serve as tags to detect the cancer cells. The tag sequenceis patient specific, and in fact it may change in the same patientbecause of clonal evolution. To define the sequence of the T or B cellreceptor from the leukemic cells for a patient the diagnostic leukemiasample that is usually highly enriched for the leukemic clone is used.For example, T and/or B cell receptor DNA is amplified from a diagnosticsample, and the product is run on a gel which can separate DNA based onsize sometime referred to as “spectratyping”); or alternativelyheteroduplex analysis can be done. A large degree of skewing of theobserved size distribution indicates monoclonal expansion, which maythen be confirmed by sequencing a sample from the skewed separationpeak. Without such subsequent sequencing, it is often difficult todetermine whether such skewing has monoclonal or polyclonal origins,e.g. Van Dongen et al, U.S. patent publication 2006/0234234.

Once the sequence tag is identified, real time PCR using Taqman probescan be used to monitor the level of that sequence. The NDN region isusually not long enough to encompass the PCR primers and the detectionoligonucleotide. Therefore typically PCR primers complementary to the Vand J regions and a Taqman probe that include some of the NDN bases ofthe leukemic clone are used. The primers provide some of thespecificity, as they amplify only a fraction of the entire repertoire.The specificity to the particular clonotype is provided by thehybridization of Taqman probe. Therefore the assay sensitivity isusually not as good as in a typical PCR (e.g., BCR-ABL) where the primerpair (with or without the Taqman probe) provides the specificity. It wasshown that the sensitivity can be as high as 10⁻⁵ for some sequences butcan be significantly worse depending on the hybridization specificityprovided by the Taqman probe whose sequence is complementary to at leastpart of the NDN region. Given the low sensitivity for some probes theassay may not work for any of the rearrangements in a particularpatient. The issue of clone evolution has also been raised previouslyfurther reducing the likelihood of detecting low level leukemia. Inaddition this technique is cumbersome requiring the generation ofpatient-specific Taqman probes as well as template to be used asstandards. These patient-specific standards need to be used at each timethe patient sample is to be tested. The inconsistency of the sensitivityamong patients, the cumbersome nature, and the logistical issues ofgetting appropriate controls for the assay has greatly limited its use.Therefore there is a need to generate markers that can be used forrelapse monitoring in patients with lymphoid neoplasms. In someembodiments the invention disclosed herein enables a very general,sensitive, and specific set of markers to be developed to managepatients with lymphoid cancers using immune cell sequencing.

It would be advantageous for many fields, including particularly theautoimmune and lymphoid cancer fields, if there were available assaysfor assessing clonotype profiles of individuals that were more sensitiveand comprehensive than current techniques and that were generallyapplicable without the need of manufacturing individualized reagents

SUMMARY OF THE INVENTION

The present invention is directed to methods for using sequence-basedprofiles of immune repertoires, or clonotype profiles, to detect andmonitor disease or non-disease conditions. The invention is exemplifiedin a number of implementations and applications, some of which aresummarized below and throughout the specification.

In one aspect the invention is directed to a method of monitoring adisease comprising the steps of (a) identifying one or morepatient-specific clonotypes correlated with a disease by determining aclonotype profile from a sample of lymphocytes in a disease-relatedtissue, the sample comprising a repertoire of clonotypes from thedisease-related tissue; (b) determining a clonotype profile from asample of peripheral blood cells to identify a presence, absence and/orlevel of the one or more patient-specific clonotypes correlated with thedisease, such peripheral blood sample comprising a repertoire ofclonotypes; and (c) repeating step (b) to monitor the disease orcondition in the patient. In one embodiment, the step of identifyingfurther includes determining a clonotype profile from a sample oflymphocytes in a non-disease-related tissue in the same patient andcomparing such clonotype profile with that from said disease-relatedtissue to identify said one or more patient-specific clonotypes.Diseases that can be monitored include, but are not limited to, lymphoidproliferative disorders, solid tumors, infectious diseases, andautoimmune diseases. The size of repertoire may vary widely depending onparticular applications; but in one embodiment, a repertoire includesevery clonotype in a sample from an individual present at a frequency of0.01 percent or greater with a probability of ninety-nine percent. Inother embodiments, a repertoire includes every clonotype in a samplefrom an individual present at a frequency of 0.001 percent or greaterwith a probability of ninety-nine percent.

In another aspect the invention is directed to a method for monitoring adisease comprising the steps of A method of monitoring a disease in apatient, the method comprising the steps of: (a) determining a clonotypeprofile from a sample of lymphocytes of an individual afflicted with adisease and a clonotype profile from the same individual from a sampleof lymphocytes enriched on the basis of cell surface markers associatedwith the disease to identify one or more patient-specific clonotypescorrelated with the disease, wherein each of the samples comprises arepertoire of clonotypes of the enriched and non-enriched lymphocytepopulations; (b) determining a level of each of the one or morepatient-specific clonotypes in a clonotype profile from a sample ofperipheral blood cells, such sample having a defined volume andcomprising a repertoire of clonotypes thereof; and (c) repeating step(b) to monitor the disease or condition in the patient.

In further embodiments of the above methods, the respective steps ofdetermining a repertoire from a sample of peripheral blood cells furthercomprises including as one or more patient-specific clonotypes anypreviously unrecorded clonotypes that are phylogenic clonotypes of theone or more patient-specific clonotypes. Whenever the disease is alymphoid proliferative disorder, the one or more patient-specificclonotypes of such embodiments may further include additionalcancer-related mutations and genetic rearrangements, such as V regionreplacements (described more fully below) readily identified by methodsof the invention.

In another aspect, the invention further provides a method ofsimultaneously measuring lymphocyte numbers and clonotype expressionlevels in a sample comprising the steps of: (i) obtaining from anindividual a sample comprising T cells and/or B cells; (ii) sequencingspatially isolated individual molecules derived from genomic DNA of saidcells, such spatially isolated individual molecules comprising a numberof clonotypes corresponding to a number of lymphocytes in the sample;(iii) sequencing spatially isolated individual molecules derived fromRNA of said cells, such spatially isolated individual moleculescomprising numbers of clonotypes corresponding to expression levelsthereof in the lymphocytes of the sample; and (iv) determining clonotypeexpression levels in lymphocytes of the sample by comparing for eachclonotype the number determined from isolated individual moleculesderived from genomic DNA of said cells and the number determined fromisolated individual molecules derived from RNA of said cells.

The invention overcomes several deficiencies in the prior art byproviding, among other advantages, sequence-based methods for measuringwith much greater sensitivity clonotypes correlated with disease orhealth conditions. The invention further provides such assays in ageneral format applicable to any patient without the need formanufacturing individualized or patient-specific reagents. Such advanceshave particularly useful applications in the areas of autoimmunity andlymphoid cancers. In the latter area, the invention further providesassay and monitoring methods that are capable of detecting and trackingnot only very low levels of disease-correlated clonotypes but also suchclonotypes that have undergone modifications that would escape detectionby prior methodologies. This latter feature is of tremendous value, forexample, in monitoring minimal residual disease in lymphoid cancers.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention is obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A is a flow diagram of an embodiment of a method of the providedinvention for determining clonotype profiles.

FIG. 1B illustrates the relative distribution of somatic mutations thatoccurs in immunoglobulins produced by B cells.

FIGS. 2A-2B show a two-staged PCR scheme for amplifying TCRβ genes.

FIG. 3A illustrates a PCR product that was amplified using the scheme ofFIGS. 2A-2B, which is going to undergo a secondary PCR to add bridgeamplification and sequencing primer binding sites for Solexa-basedsequencing. FIG. 3B illustrates details of one embodiment of determininga nucleotide sequence of the PCR product of FIG. 3A. FIG. 3C illustratesdetails of another embodiment of determining a nucleotide sequence ofthe PCR product of FIG. 3A.

FIG. 4A illustrates a PCR scheme for generating three sequencingtemplates from an IgH chain in a single reaction. FIGS. 4B-4Cillustrates a PCR scheme for generating three sequencing templates froman IgH chain in three separate reactions after which the resultingamplicons are combined for a secondary PCR to add P5 and P7 primerbinding sites. FIG. 4D illustrates the locations of sequence readsgenerated for an IgH chain. FIG. 4E illustrates the use of the codonstructure of V and J regions to improve base calls in the NDN region.

FIG. 5 shows data evidencing the reproducibility of multiplex PCRs ofthe invention.

FIG. 6 shows data that demonstrate that multiplex PCRs of the inventionintroduce minimal amplification bias.

FIG. 7A shows the log 10 of the frequency of each clonotype in the twoduplicate samples using Accuprime and cDNA corresponding to 500 ng ofRNA as input template.

FIG. 7B depicts the log 10 of the frequency of each clonotype using cDNAcorresponding to 500 ng of RNA as input template and Accuprime (X axis)or High fidelity Taq (Y axis).

FIG. 7C shows the log 10 of the frequency of each clonotype using cDNAcorresponding to 50 ng of RNA as input template and Accuprime (X axis)or High fidelity Taq (Y axis).

FIGS. 8A and 8B show data from IgH amplification from genomic DNA.

FIG. 9 shows data indicating that multiplex amplifications in accordancewith the invention have minimal amplification bias.

FIG. 10 shows data comparing clonotypes of two individuals.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of this invention utilizes next generation sequencingtechnologies to evaluate the levels of TCR or BCR rearrangements in apopulation of lymphocytes. These sequencing technologies can obtain 1million or more reads from a sample at a reasonable cost. A clonotypepresent at a frequency of 1/1,000,000 or lower can still be detected ina specific manner using these technologies. Multiplex amplification toamplify all the different types of sequences of a specific portion ofgene or transcript can be accomplished from a sample from blood or bonemarrow DNA. For example, to amplify IgH sequences, several primerscomplementary to all the known V segments and alleles can be used alongwith several primers complementary to all the J segments and alleles.FIG. 1A illustrates steps of such a method for an embodiment employingone class of DNA sequencers (e.g. Solexa sequencing-by-synthesis, asdescribed below) for profiling clonotypes of a sample. A samplecontaining B cells or T cells is obtained (100) after which DNA or RNAis extracted and amplified (102) in a reaction that preferentiallyamplifies clonotypes and attaches terminal sequences for subsequentamplification and sequencing. Individual molecules of the amplifiedclonotypes are randomly distributed on a solid surface (104), such as, aglass surface, which has been configured to permit a second in situamplification to produce clonal populations (or polonies) of eachindividual molecule (106). The molecules of each polony are thensequenced (108), for example, using a sequencing-by-synthesis technique,after which the types and abundances of the sequences are tabulated toform a clonotype profile (110), or equivalently a repertoire profile.The method can be performed with little amplification bias among thedifferent sequences. RNA from the TCRβ and IgH genes can be amplifiedwith only small differences in the efficiencies of the different Vprimers, thereby validating the possibility of doing the same from DNA.This scheme can ameliorate problems for the real time readout fordetection of low level TCR and/or BCR rearrangements.

Sensitivity is determined by counting statistics (that is, sensitivityis increased by increasing cell and sequencing sample sizes) andequivalent amplification (that is, clonotypes having varied sequencesmay be amplified without significant bias in a multiplex amplificationreaction, such as a PCR, as illustrated below). Since sensitivity isultimately limited by counting statistics, to obtain more sensitivityone can simply obtain more cells (i.e. larger samples) and moresequencing reads. With sufficient sequencing reads the sensitivity islimited by the number of lymphocytes in the sample. In contrast,sensitivity for the real time PCR assay is limited by background.Moreover a patient's specific clones can be determined by sequencing adiagnostic leukemia or lymphoma sample. Once the clonotype isdetermined, its level can be determined in samples at subsequent timepoints. In some preferred embodiments there is no requirement for apatient-specific probes or primers or the utilization ofpatient-specific templates to be run as standards. Insteadpatient-specific clones are followed by storing the data regarding therelevant sequences for each patient, and the same assay works for allpatients.

In general, some embodiments of the invention include methods forapplying nucleic acid sequencing techniques to the task of monitoringthe repertoire of adaptive immunity cells for profiling the immunesystem. The profiles of the immune system generated can be used fordiagnosis of diseases and disorders, and for diagnosis of states ofdiseases and disorders. The methods of immune profiling of the providedinvention can be used in monitoring diseases and disorders and assessingtreatment of diseases and disorders. The diseases and disorders that themethods of the provided invention can be applied to include autoimmunedisease, including systemic lupus erythematosus (SLE), multiplesclerosis (MS), rheumatoid arthritis (RA), and ankylosing spondylitis(AS). The methods of the provided invention can be applied to thediagnosis, monitoring, and treatment of transplant rejection and immuneaging. Furthermore, the methods of immune profiling of the providedinvention can be used for diagnosing, monitoring, and treating otherdiseases related to the immune system, including cancer and infectiousdisease.

Sequencing individual amplified molecules can distinguish differentsequences and hence has the sensitivity to detect quantitative changesin clonal expansion. In general, in one embodiment of the providedinvention, a method for determining a profile of recombined DNAsequences in T-cells and/or B-cells is provided. The method can comprisesteps including isolating samples from a subject, one or more rounds ofnucleic acid amplification, spatially isolating individual nucleicacids, and sequencing nucleic acids. The nucleic acids can be DNA orRNA. The recombined DNA sequences in T-cells and/or B-cells can betermed clonotypes.

In one aspect, a method for determining one or more correlatingclonotypes in a subject or individual is provided. In another aspect, amethod for developing an algorithm that can predict one or morecorrelating clonotypes in any sample from a subject with a disease isprovided. In another aspect, a method for discovering one or morecorrelating clonotypes for an individual using an algorithm that canpredict one or more correlating clonotypes in any sample from a subjectis provided. In another aspect, a method for generating an algorithmthat calculates a disease activity score is provided. In another aspect,a method for monitoring the disease state of an individual is provided.

T and B cell repertoire profiling can be of value for diseases withinflammatory aspects. This inflammation is often due to autoimmuneand/or hypersensitivity reaction. These diseases include cardiovasculardisease, Alzheimer disease, and pre-eclampsia. Inflammation has alsobeen associated with abnormal metabolic states including obesity anddiabetes. Other inflammation related diseases exist. In one aspect ofthe invention, a segment of recombined B cell nucleic acid is amplifiedby a PCR with a plurality of forward primers or a plurality of reverseprimers to generate a nested set of templates (see FIGS. 4A and 4B andtheir descriptions below). Templates from such a set may be furtheramplified on a surface to form separate amplicons (e.g. by bridge PCRusing a cBot instrument, Illumina, San Diego, Calif.). Templates fromthe same nested set may be associated with one another by sequence readsgenerated at their common ends. Nested sets of templates allow asequencing chemistry with relative high error rates to be used toanalyze longer sequences than otherwise would be possible, while at thesame time maintaining high average quality scores over the entire lengthof the sequence. The nested sets also ensure that at least one sequenceread is obtained from a V region even if it has been subjected tosomatic hypermutation. In one embodiment, sequencing chemistries may beused for analyzing highly variable nucleic acids, such as IgH molecules,that have error rates no better than the following: 0.2 percent ofsequence reads contain at least one error in positions 1-50; 0.2-1.0percent of sequence reads contain at least one error in positions 51-75;0.5-1.5 percent of sequence reads contain at least one error inpositions 76-100; and 1-5 percent of sequence reads contain at least oneerror in positions 101-125. In another embodiment, sequencing primerbinding sites are positioned so that when extended they produce a seriesof sequence reads where each sequence read except the last overlaps itsimmediately adjacent downstream primer binding site and/or sequenceread, thereby providing continuous sequence coverage with higher qualityscores than would be possible if a single long template were used togenerate a single long sequence read.

I. Further Aspects and Embodiments

Further aspects and embodiments of the invention include the following:A method for determining a profile of recombined DNA sequences inT-cells and/or B-cells is provided comprising: obtaining a sample from asubject comprising T-cells and/or B-cells, spatially isolatingindividual molecules of genomic DNA from said cells; sequencing saidspatially isolated individual molecules of genomic DNA, and determiningthe levels of different sequences from said sample to generate saidprofile of recombined DNA sequences. A method for determining a profileof recombined DNA sequences in T-cells and/or B-cells is providedcomprising: obtaining a sample from a subject comprising T-cells and/orB-cells, spatially isolating individual molecules of genomic DNA fromsaid cells, amplifying said individual molecules of genomic DNA,sequencing said amplified DNA, and determining the levels of differentsequences from said sample to generate said profile of recombined DNAsequences. A method for determining a profile of recombined DNAsequences in T-cells and/or B-cells is provided comprising: obtaining asample from a subject comprising T-cells and/or B-cells, amplifyinggenomic DNA from said cells, spatially isolating individual molecules ofsaid amplified DNA, sequencing said spatially isolated individualmolecules of amplified DNA; and determining the levels of differentsequences from said sample to generate said profile of recombined DNAsequences. A method for determining a profile of recombined DNAsequences in T-cells and/or B-cells is provided comprising: obtaining asample from a subject comprising T-cells and/or B-cells, amplifyinggenomic DNA from said cells, spatially isolating individual molecules ofsaid amplified DNA, re-amplifying said amplified DNA molecules,sequencing said re-amplified DNA molecules, and determining the levelsof different sequences from said sample to generate said profile ofrecombined DNA sequences. A method for determining a profile ofsequences of recombined DNA in T-cells and/or B-cells is providedcomprising: obtaining a sample from a subject comprising T-cells and/orB-cells, reverse transcribing RNA from said cells to form cDNA,spatially isolating individual molecules of said cDNA, optionallyre-amplifying said spatially isolated individual molecules of cDNA,sequencing said cDNA and/or re-amplified cDNA; and determining thelevels of different sequences from said sample to generate said profileof recombined DNA sequences. A method for determining a profile ofrecombined DNA sequences in T-cells and/or B-cells is providedcomprising: obtaining a sample from a subject comprising T-cells and/orB-cells; spatially isolating individual cells in said sample, sequencingindividual molecules of nucleic from said cells; and determining thelevels of different sequences from said sample to generate said profileof recombined DNA sequences. In one embodiment, said amplifying and/orre-amplifying comprises PCR, multiplex PCR, TMA, NASBA, or LAMP. Inanother embodiment, said spatially isolating comprises separating saidDNA or cDNA in two dimensions on a solid support, separating said DNA orcDNA in three dimensions in a solution with micelles, or separatingmolecules using micro-reaction chambers. In another embodiment, saidamplifying and/or re-amplifying is by growth of bacteria harboringsubcloned DNA or cDNA, amplification of DNA or cDNA on a slide, oramplification of DNA or cDNA on a bead. In another embodiment, saidsequencing comprises dideoxy sequencing. In another embodiment, saidsequencing comprises sequencing by synthesis using reversibly terminatedlabeled nucleotides. In another embodiment, said sequencing comprisesdetection of pyrophosphate release on nucleotide incorporation. Inanother embodiment, said sequencing comprises allele specifichybridization to a library of labeled oligonucleotide probes. In anotherembodiment, said sequencing comprises sequencing by synthesis usingallele specific hybridization to a library of labeled oligonucleotideprobes followed by ligation of said probes. In another embodiment, saidsequencing comprises real time monitoring of the incorporation oflabeled nucleotides during a polymerization step. In another embodiment,said recombined DNA sequences comprise T-cell receptor genes and/orimmunoglobulin genes. In another embodiment, said sequencing comprisessequencing a subset of the full clonal sequences of immunoglobulinand/or T-cell receptor genes. In another embodiment, said subset of thefull clonal sequence comprises the V-D junction, D-J junction of animmunoglobulin or T-cell receptor gene, the full variable region of animmunoglobulin or T-cell receptor gene, the antigen recognition region,or the complementarity determining region 3 (CDR3). In anotherembodiment, said T-cell receptor genes comprise T-cell receptor β genes.In another embodiment, said immunoglobulin genes comprise immunoglobulinheavy genes. In another embodiment, said amplifying or re-amplifyingcomprises a plurality of primers complementary to V segments and oneprimer complementary to a C segment. In another embodiment, saidamplifying or re-amplifying comprises a plurality of primerscomplementary to V segments and a plurality of primers complementary toC segments. In another embodiment, said plurality of primerscomplementary to V segments comprises at least three different primersfor each V segment and the plurality of primers complementary to Csegments comprises at least 1, at least 2, at least 3, at least 4, atleast 5, or at least 6 primers. In another embodiment, said T- orB-cells are subsets of the total T and B cells. In another embodiment,said subset of T-cells are CD4+, CD8+ cells, or CD27 high cells. Inanother embodiment, said sample comprises at least 100,000, at least500,000, at least 750,000, of at least 1,000,000 T-cells. In anotherembodiment, said sequencing comprises at least 1000 reads per run, atleast 10,000 reads per run, at least 100,000 reads per run, or at least1,000,000 reads per run. In another embodiment, said sequencingcomprises generating about 30 bp, about 40 bp, about 50 bp, about 60 bp,about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110, or about120 bp per read. In another embodiment, said sample is taken when thesubject is at a flare state of an autoimmune disease. In anotherembodiment, said sample is taken from a subject having or suspected ofhaving systemic lupus erythematosus. In another aspect, a method fordetermining one or more correlating clonotypes in a subject is providedcomprising: generating one or more clonotype profiles by nucleic acidsequencing individual, spatially isolated molecules from at least onesample from the subject, wherein the at least one sample is related to afirst state of the disease, and determining one or more correlatingclonotypes in the subject based on the one or more clonotype profiles.In one embodiment, said at least one sample is from a tissue affected bythe disease. In another embodiment, said determination of one or morecorrelating clonotypes comprises comparing clonotype profiles from atleast two samples. In another embodiment, the first state of the diseaseis a peak state of the disease. In another embodiment, said one or morecorrelating clonotypes are present in the peak state of the disease. Inanother embodiment, said one or more correlating clonotypes are absentin the peak state of the disease. In another embodiment, said one ormore correlating clonotypes are high in the peak state of the disease.In another embodiment, said one or more correlating clonotypes are lowin the peak state of the disease. In another embodiment, said samplecomprises T-cells and/or B-cells. In another embodiment, said T-cellsand/or B-cells comprise a subset of T-cells and/or B-cells. In anotherembodiment, said subset of T-cells and/or B-cells are enriched byinteraction with a marker. In another embodiment, said marker is a cellsurface marker on the subset of T-cells and/or B-cells. In anotherembodiment, said subset of T-cells and/or B-cells interact with anantigen specifically present in the disease. In another embodiment, thedisease is systemic lupus erythematosus or multiple sclerosis. Inanother aspect, a method for developing an algorithm that can predictone or more correlating clonotypes in any sample from a subject with adisease is provided comprising: a) generating a plurality of clonotypeprofiles from a set of samples, wherein the samples are relevant to thedisease, b) identifying one or more correlating clonotypes from the setof samples, c) using sequence parameters and/or functional data from oneor more correlating clonotypes identified in b) to develop the algorithmthat can predict correlating clonotypes in any sample from a subjectwith the disease. In one embodiment, the set of samples are taken fromone or more tissues affected by the disease. In another embodiment, saididentification of one or more correlating clonotypes comprises comparingclonotype profiles from at least two samples. In another embodiment,said functional data include binding ability of markers on T-cell and/orB-cell surface or interaction with antigen by a T-cell or B-cell. Inanother embodiment, said sequence parameters comprise nucleic acidsequence and predicted amino acid sequence. In another embodiment, thesamples are from one or more individuals at a peak stage of the disease.In another embodiment, said one or more correlating clonotypes arepresent in the peak state of the disease. In another embodiment, saidone or more correlating clonotypes are at a high level in the peak stateof the disease. In another embodiment, said one or more correlatingclonotypes are at a low level in the peak state of the disease. Inanother embodiment, the one or more correlating clonotypes are absent atthe peak state of the disease. In another embodiment, the disease issystemic lupus erythematosus or multiple sclerosis. In anotherembodiment, a method for discovering one or more correlating clonotypesfor an individual is provided, comprising inputting a clonotype profilefrom a sample from the individual into an algorithm, and using thealgorithm to determine one or more correlating clonotypes for theindividual. In one embodiment, the algorithm is an algorithm that canpredict one or more correlating clonotypes in any sample from a subjectwith a disease is provided comprising, said algorithm being developedby: a) generating a plurality of clonotype profiles from a set ofsamples, wherein the samples are relevant to the disease, b) identifyingone or more correlating clonotypes from the set of samples, c) usingsequence parameters and/or functional data from one or more correlatingclonotypes identified in b) to develop an algorithm that can predictcorrelating clonotypes in any sample from a subject with the disease. Inone embodiment, said sample is at taken at a peak state of disease. Inanother embodiment, the sample is taken from disease affected tissue. Inanother aspect, a method for generating an algorithm that calculates adisease activity score is provided comprising: developing an algorithmthat uses a set of factors to combine levels of correlating clonotypesinto a disease activity score, comparing the disease activity score toclinical data regarding the disease state, and optimizing the factors inorder to maximize the correlation between clinical data and the diseaseactivity score. In one embodiment, method for monitoring the diseasestate of an individual is provided comprising: a) determining aclonotype profile from a sample from the individual, b) inputting theclonotype profile information from a) into an algorithm that calculatesa disease activity score, wherein is algorithm is generated bydeveloping an algorithm that uses a set of factors to combine levels ofcorrelating clonotypes into a disease activity score, comparing thedisease activity score to clinical data regarding the disease state, andoptimizing the factors in order to maximize the correlation betweenclinical data and the disease activity score, and c) using the algorithmthat calculates a disease activity score to generate a score predictiveof the disease state of the individual. In another embodiment, themethod for monitoring the disease state of an individual furthercomprises determining one or more correlating clonotypes in theindividual, and inputting information the one or more correlatingclonotypes into the algorithm. In another embodiment, said determiningone or more correlating clonotypes in the individual comprises a)generating one or more clonotype profiles by nucleic acid sequencingindividual, spatially isolated molecules from at least one sample fromthe subject, wherein the at least one sample is related to a first stateof the disease, and b) determining one or more correlating clonotypes inthe subject based on the one or more clonotype profiles. In anotherembodiment, said determining one or more correlating clonotypes in theindividual comprises a) inputting a clonotype profile from a sample fromthe individual into an algorithm that can predict one or morecorrelating clonotypes, wherein said algorithm that can predict one ormore correlating clonotypes is developed by i) generating a plurality ofclonotype profiles from a set of samples, wherein the samples arerelevant to the disease, ii) identifying one or more correlatingclonotypes from the set of samples, iii) using sequence parametersand/or functional data from one or more correlating clonotypesidentified in ii) to develop the algorithm that can predict correlatingclonotypes in any sample from a subject with the disease, and c) usingthe algorithm that can predict one or more correlating clonotypes todetermine one or more correlating clonotypes for the individual. Inanother embodiment, the disease is systemic lupus erythematosus ormultiple sclerosis. In another aspect, a method of determining one ormore correlating T or B cell clonotypes is provided comprising: a)dividing a sample of cells from a subject into at least two samples, b)generating one or more clonotype profiles by nucleic acid sequencingindividually spatially isolated molecules from one of the samples ofcells from the subject, c) enriching another sample of cells from thissubject based on at least one molecular parameter of the cells, d)generating a one of more clonotype profiles by nucleic acid sequencingindividually spatially isolated molecules from the enriched sample ofthe subject, and e) identifying at least one clonotype based onclonotypes whose abundance within the sample has been altered betweenthe enriched sample and the unenriched sample. In another embodiment,the molecular parameter is a cell surface marker. In another embodiment,the enrichment is done by capturing cells using a solid phaseimmobilized affinity marker. In another embodiment, the solid surface isa set of beads. In another embodiment, the solid surface is a column. Inanother embodiment, the marker is labeled using a fluorescent moiety. Inanother embodiment, the enrichment is accomplished by flow cytometryusing the fluorescent label. In another embodiment, the cells are Blymphocytes and the enrichment is done using antigens that bind the Bcell receptor. In another embodiment, the enrichment is done throughcapture a solid surface on which antigens are immobilized. In anotherembodiment, the antigen is used to label the B lymphocytes and theenrichment is accomplished using flow cytometry using this label. Inanother embodiment, the cells are T lymphocytes and the enrichment isdone using a method that allows the T cells that react to a specificantigen to be labeled and enriched using flow cytometry. In anotherembodiment, the T cells are labeled using the intracellular cytokinestaining method. In another embodiment, the T cells are labeled usingthe cytokine capture method. In another embodiment, the molecularparameter the B cell receptor that is capable of binding at least oneantigen that is specific to a pathogen. In another embodiment, themolecular parameter is the T cell receptor that is capable of binding atleast on antigen that is specific to a pathogen. In another embodiment,the sample is taken from a patient that has been exposed to a pathogenat a first time point. In another aspect a method for determining a setof clonotypes in an individual that correlate with an immune reaction toa pathogen is provided comprising: a) dividing a sample of cells from asubject into at least two samples b) generating one or more clonotypeprofiles by nucleic acid sequencing individually spatially isolatedmolecules from one sample of cells from the subject, c) enrichinganother sample of cells from this subject based on the cells ability tobind at least one antigen from the pathogen d) generating a one of moreclonotype profiles by nucleic acid sequencing individually spatiallyisolated molecules from the enriched sample of the subject and, e)identifying at least one correlating clonotype based on clonotypes whoseabundance within the sample has been altered between the enriched sampleand the unenriched sample. In another aspect a method for determining aset of clonotypes in an individual that correlate with an immunereaction to a tumor is provided comprising: a) dividing a sample ofcells from a subject into at least two samples b) generating one or moreclonotype profiles by nucleic acid sequencing individually spatiallyisolated molecules from one sample of cells from the subject c)enriching another sample of cells from this subject based on the cellsability to bind at least one autoantigen present in the tumor d)generating a one of more clonotype profiles by nucleic acid sequencingindividually spatially isolated molecules from the enriched sample ofthe subject e) identifying at least one correlating clonotype based onclonotypes whose abundance within the sample has been altered betweenthe enriched sample and the unenriched sample. In another embodiment,the levels of correlating clonotypes are used to assess the risk thatthe individual has a tumor. In another embodiment, the antigens areknown to be present in a tumor that has already occurred in thatindividual and the correlating clonotypes are used to assess the risk oftumor recurrence. In another embodiment, the antigens are known to bepresent in tumors in other individuals and the clonotypes are used toassess the risk of cancer in a patient who has not had a tumor detectedpreviously. In another aspect a method for determining a set ofclonotypes in an individual that correlate with an immune reaction to amaterials released in the bloodstream by damage to an organ is providedcomprising: a) dividing a sample of cells from a subject into at leasttwo samples b) generating one or more clonotype profiles by nucleic acidsequencing individually spatially isolated molecules from one sample ofcells from the subject c) enriching another sample of cells from thissubject based on the cells ability to bind at least one autoantigenpresent in the damaged organs d) generating a one of more clonotypeprofiles by nucleic acid sequencing individually spatially isolatedmolecules from the enriched sample of the subject and e) identifying atleast one correlating clonotype based on clonotypes whose abundancewithin the sample has been altered between the enriched sample and theunenriched sample. In another embodiment, the levels of correlatingclonotypes are used to assess the risk that the individual has organdamage. In another aspect a method for determining a set of clonotypesin an individual that correlate with an immune reaction to a therapeuticagent is provided comprising: a) dividing a sample of cells from asubject into at least two samples b) generating one or more clonotypeprofiles by nucleic acid sequencing individually spatially isolatedmolecules from one sample of cells from the subject c) enriching anothersample of cells from this subject based on the cells ability to bind atleast one antigen contained in the therapeutic agent d) generating a oneof more clonotype profiles by nucleic acid sequencing individuallyspatially isolated molecules from the enriched sample of the subject ande) identifying at least one correlating clonotype based on clonotypeswhose abundance within the sample has been altered between the enrichedsample and the unenriched sample. In another embodiment, the levels ofcorrelating clonotypes are used to assess the risk that the individualis exhibiting hypersensitivity to a therapeutic agent. In another aspecta method for determining a set of clonotypes in an individual thatcorrelate with an immune reaction to a arterial plaque is providedcomprising: a) dividing a sample of cells from a subject into at leasttwo samples b) generating one or more clonotype profiles by nucleic acidsequencing individually spatially isolated molecules from one sample ofcells from the subject c) enriching another sample of cells from thissubject based on the cells ability to bind at least one antigen presentin arterial plaque d) generating a one of more clonotype profiles bynucleic acid sequencing individually spatially isolated molecules fromthe enriched sample of the subject and e) identifying at least onecorrelating clonotype based on clonotypes whose abundance within thesample has been altered between the enriched sample and the unenrichedsample In another embodiment, the levels of correlating clonotypes areused to assess the risk that the individual has a cardiovasculardisease. In another embodiment, the levels of correlating clonotypes areused to assess the risk that arterial plaque is unstable. In anotheraspect a method for determining a sequence identifier found in cellsinvolved in a lymphoid neoplasm is described comprising: a) obtaining asample of cells from the affected individual in which the cancerouscells are known to exist b) generating one or more clonotype profilesrelated to at least one immune cell genomic rearrangement by nucleicacid sequencing individually spatially isolated molecules from the cellsin the sample c) identifying the sequence identifier as the sequence ofthe clonotype associated with the tumor. In another embodiment, thesample is from the bone marrow of the patient. In another embodiment,the sample is from the blood of the patient. In another embodiment, thesample is from a biopsy of a solid lymphoid tumor. In anotherembodiment, the immune cell genomic rearrangement is a VDJ rearrangementof IgH in a B cell. In another embodiment, the immune cell genomicrearrangement is a DJ rearrangement of IgH in a B cell. In anotherembodiment, the immune cell genomic rearrangement is a VJ rearrangementof IgK in a B cell. In another embodiment, the immune cell genomicrearrangement is a VJ rearrangement of IgL in a B cell. In anotherembodiment, the immune cell genomic rearrangement is a VDJ rearrangementof TCR β in a T cell. In another embodiment, the immune cell genomicrearrangement is a DJ rearrangement of TCR β in a T cell. In anotherembodiment, the immune cell genomic rearrangement is a VJ rearrangementof TCR α in a T cell. In another embodiment, the immune cell genomicrearrangement is a VJ rearrangement of TCR λ in a T cell. In anotherembodiment, the immune cell genomic rearrangement is a VDJ rearrangementof TCR δ in a T cell. In another embodiment, the immune cell genomicrearrangement is a VD rearrangement of TCR δ in a T cell. In anotherembodiment, the immune cell genomic rearrangement is a translocation ofa J segment of IgH to another region of the genome. In anotherembodiment, the immune cell genomic rearrangement is a translocation ofany J segment to another region of the genome. In another embodiment,the identification of the tumor associated clonotype is done byclonotype frequency. In another embodiment, the identification of thetumor associated clonotype is done by clonotype frequency. In anotherembodiment, the identification of the tumor associated clonotype is doneby the detection of cross lineage rearrangement. In another embodiment,the identification of the tumor associated clonotype is done byidentifying nonfunctional rearrangements \ In another embodiment, theidentification of the tumor associated clonotype is done by associatingcell clonotypes with at least one molecular marker associated with thetumor. In another aspect a method of determining the levels ofcirculating lymphoid tumor cells within an individual whose tumor hasbeen associated with a unique sequence identifier at a first time pointinvolving is described comprising: a) obtaining a sample of cells fromthe patient b) generating one or more clonotype profiles related to atleast one immune cell genomic rearrangement by nucleic acid sequencingindividually spatially isolated molecules from the cells in the samplec) determining the levels of tumor cells from the level of theclonotypes associated with the sequence identifier. In another aspect amethod of determining the levels of circulating lymphoid tumor cellswithin an individual whose tumor has been associated with a uniquesequence identifier at a first time point involving is describedcomprising: a) obtaining a sample of cells from the patient b) enrichingthe cells based on at least one molecular marker c) generating one ormore clonotype profiles related to at least one immune cell genomicrearrangement by nucleic acid sequencing individually spatially isolatedmolecules from the cells in the sample d) determining the levels oftumor cells from the level of the clonotypes associated with thesequence identifier. In another embodiment, the sample is a bloodsample. In another embodiment, the sample is a bone marrow sample. Inanother embodiment, the sample is a lymph sample. In another embodiment,the sample is a tissue sample. In another embodiment, the cells arelabeled fluorescently and enriched using flow cytometry. In anotherembodiment, the cells are enriched through binding to a solid support.In another embodiment, the clonotype is defined to be the clonotype thatcontains the unique sequence identifier. In another embodiment, theclonotypes are determined to be those clonotypes that are likely to haveresulted from mutations and rearrangements to the sequence identifier.In another embodiment, the levels of circulating tumor cells and/or thechange in the levels of the circulating tumor cells are used in analgorithm to produce a score that correlates with the risk of having aclinical tumor recurrence. In another embodiment, the levels ofcirculating tumor cells and/or the change in the levels of thecirculating tumor cells are used to make a treatment decision.

II. Methods of Determining Clonotype Profiles

The methods of the invention can be used to generate profiles ofrecombined DNA sequences, or clonotypes, in a sample from a subject. Inone embodiment, a method for determining a profile of recombined DNAsequences in T-cells and/or B-cells is provided including obtaining asample from a subject comprising T-cells and/or B-cells, isolatingindividual molecules of genomic DNA from said cells, sequencing theisolated individual molecules of genomic DNA, and determining the levelsof different sequences from the sample to generate said profile ofrecombined DNA sequences.

In another embodiment, a method for determining a profile of recombinedDNA sequences in T-cells and/or B-cells is provided including obtaininga sample from a subject comprising T-cells and/or B-cells, isolatingindividual molecules of genomic DNA from the cells, amplifying theindividual molecules of genomic DNA, sequencing the amplified DNA, anddetermining the levels of different sequences from the sample togenerate said profile of recombined DNA sequences.

In another embodiment, a method for determining a profile of recombinedDNA sequences in T-cells and/or B-cells is provided including obtaininga sample from a subject comprising T-cells and/or B-cells, amplifyinggenomic DNA from the cells, isolating individual molecules of theamplified DNA, sequencing the isolated individual molecules of amplifiedDNA, and determining the levels of different sequences from the sampleto generate the profile of recombined DNA sequences.

In another embodiment, a method for determining a profile of recombinedDNA sequences in T-cells and/or B-cells is provided including obtaininga sample from a subject including T-cells and/or B-cells, amplifyinggenomic DNA from the cells, isolating individual molecules of theamplified DNA, re-amplifying the amplified DNA molecules, sequencing there-amplified DNA molecules, and determining the levels of differentsequences from the sample to generate the profile of recombined DNAsequences.

In another embodiment, a method for determining a profile of sequencesof recombined DNA in T-cells and/or B-cells is provided includingobtaining a sample from a subject comprising T-cells and/or B-cells,isolating RNA from said sample, reverse transcribing the RNA from saidcells to form cDNA, isolating individual molecules of said cDNA,optionally re-amplifying said cDNA, sequencing said isolated individualmolecules of said cDNA or re-amplified DNA, and determining the levelsof different sequences from said sample to generate said profile ofrecombined DNA sequences.

In another embodiment, a method for determining a profile of sequencesof recombined DNA in T-cells and/or B-cells is provided includingobtaining a sample from a subject including T-cells and/or B-cells,isolating individual molecules of RNA from said sample, sequencing theindividual molecules of RNA, and determining the levels of differentsequences from said sample to generate the profile of recombined DNAsequences.

Subjects and Samples

The methods of the provided invention can use samples from subjects orindividuals (e.g., patients). The subject can be a patient, for example,a patient with an autoimmune disease. The subject can be a patient withan infectious disease or cancer, such as a leukemia or a lymphoma. Thesubject can be a mammal, for example, a human. The subject can be maleor female. The subject can be an infant, a child, or an adult. In someembodiments the subject is no longer living. In some embodiments thesubject is alive. The subject can be an individual who was exposed to abiologic weapon.

The subject could also be a non-human animal. The non-human animal couldbe a domestic pet or a farm animal. The non-human animal could be a dog,cat, cow, horse, goat, or pig. The non-human animal could be a clonedanimal. The non-human animal could be involved in the production ofpharmaceuticals.

Samples used in the methods of the provided invention can include, forexample, a bodily fluid from a subject, including amniotic fluidsurrounding a fetus, aqueous humor, bile, blood and blood plasma,cerumen (earwax), Cowper's fluid or pre-ejaculatory fluid, chyle, chyme,female ejaculate, interstitial fluid, lymph, menses, breast milk, mucus(including snot and phlegm), pleural fluid, pus, saliva, sebum (skinoil), semen, serum, sweat, tears, urine, vaginal lubrication, vomit,water, feces, internal body fluids, including cerebrospinal fluidsurrounding the brain and the spinal cord, synovial fluid surroundingbone joints, intracellular fluid is the fluid inside cells, and vitreoushumour the fluids in the eyeball. In one embodiment, the sample is ablood sample. The blood sample can be about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mL.The sample can be Cerebral Spinal Fluid (CSF) when the subject hasmultiple sclerosis, synovial fluid when the subject has rheumatoidarthritis, and skin (or other organ) biopsy when the subject hassystemic lupus. In one embodiment, the clonotype can be identified fromthe available body fluid/tissue most likely to reflect pathologyfollowed by later monitoring the levels of the clonotypes form adifferent body fluid, for example, blood. Samples can also includesolvents in which biologic material has been dissolved. Samples can beanalyzed at a time when a disease is inactive. Samples can be analyzedat a time when a disease is active. Samples can be obtained at a timewhen a disease is inactive. Samples can be obtained at a time when adisease is active. The sample can be obtained by a health care provider,for example, a physician, physician assistant, nurse, veterinarian,dermatologist, rheumatologist, dentist, paramedic, or surgeon. Thesample can be obtained by a research technician. The sample can beprovided by the subject. The sample can be provided anonymously. Thesample can be provided through the mail. The sample can be provided by alaw enforcement agency or by an investigator. More than one sample froma subject can be obtained.

The sample can be a biopsy, e.g., a skin biopsy. The biopsy can be from,for example, brain, liver, lung, heart, colon, kidney, or bone marrow.Any biopsy technique used by those skilled in the art can be used forisolating a sample from a subject. For example, a biopsy can be an openbiopsy, in which general anesthesia is used. The biopsy can be a closedbiopsy, in which a smaller cut is made than in an open biopsy. Thebiopsy can be a core or incisional biopsy, in which part of the tissueis removed. The biopsy can be an excisional biopsy, in which attempts toremove an entire lesion are made. The biopsy can be a fine needleaspiration biopsy, in which a sample of tissue or fluid is removed witha needle.

The sample can be obtained from bodily material which is left behind bya subject. Such discarded material can include human waste. Discardedmaterial could also include shed skin cells, blood, teeth or hair.

The sample can include immune cells, for example, the immune cells caninclude T-cells and/or B-cells. T-cells (T lymphocytes) include, forexample, cells that express T cell receptors. T-cells include Helper Tcells (effector T cells or Th cells), cytotoxic T cells (CTLs), memory Tcells, and regulatory T cells. The sample can include a single cell insome applications (e.g., a calibration test to define relevant T cells)or more generally at least 1,000, at least 10,000, at least 100,000, atleast 250,000, at least 500,000, at least 750,000, or at least 1,000,000T-cells.

B-cells include, for example, plasma B cells, memory B cells, B1 cells,B2 cells, marginal-zone B cells, and follicular B cells. B-cells canexpress immunoglobulins (antibodies, B cell receptor). The sample caninclude a single cell in some applications (e.g., a calibration test todefine relevant B cells) or more generally at least 1,000, at least10,000, at least 100,000, at least 250,000, at least 500,000, at least750,000, or at least 1,000,000 B-cells.

The sample can include nucleic acid, for example, DNA (e.g., genomic DNAor mitochondrial DNA) or RNA (e.g., messenger RNA or microRNA). Thenucleic acid can be cell-free DNA or RNA, e.g. extracted from thecirculatory system, Vlassov et al, Curr. Mol. Med., 10: 142-165 (2010);Swamp et al, FEBS Lett., 581: 795-799 (2007). In the methods of theprovided invention, the amount of RNA or DNA from a subject that can beanalyzed includes, for example, as low as a single cell in someapplications (e.g., a calibration test) and as many as 10 million ofcells or more translating to a range of DNA of 6 pg-60 ug, and RNA ofapproximately 1 pg-10 ug.

As discussed more fully below (Definitions), a sample of lymphocytes issufficiently large so that substantially every T cell or B cell with adistinct clonotype is represented therein, thereby forming a repertoire(as the term is used herein). In one embodiment, a sample is taken thatcontains with a probability of ninety-nine percent every clonotype of apopulation present at a frequency of 0.001 percent or greater. Inanother embodiment, a sample is taken that contains with a probabilityof ninety-nine percent every clonotype of a population present at afrequency of 0.0001 percent or greater. In one embodiment, a sample of Bcells or T cells includes at least a half million cells, and in anotherembodiment such sample includes at least one million cells.

Whenever a source of material from which a sample is taken is scarce,such as, clinical study samples, or the like, DNA from the material maybe amplified by a non-biasing technique, such as whole genomeamplification (WGA), multiple displacement amplification (MDA); or liketechnique, e.g. Hawkins et al, Curr. Opin. Biotech., 13: 65-67 (2002);Dean et al, Genome Research, 11: 1095-1099 (2001); Wang et al, NucleicAcids Research, 32: e76 (2004); Hosono et al, Genome Research, 13:954-964 (2003); and the like.

Blood samples are of particular interest, especially in monitoringlymphoid neoplasms, such as lymphomas, leukemias, or the like, and maybe obtained using conventional techniques, e.g. Innis et al, editors,PCR Protocols (Academic Press, 1990); or the like. For example, whiteblood cells may be separated from blood samples using conventiontechniques, e.g. RosetteSep kit (Stem Cell Technologies, Vancouver,Canada). Blood samples may range in volume from 100 μL to 10 mL; in oneaspect, blood sample volumes are in the range of from 200 100 μL to 2mL. DNA and/or RNA may then be extracted from such blood sample usingconventional techniques for use in methods of the invention, e.g. DNeasyBlood & Tissue Kit (Qiagen, Valencia, Calif.). Optionally, subsets ofwhite blood cells, e.g. lymphocytes, may be further isolated usingconventional techniques, e.g. fluorescently activated cell sorting(FACS)(Becton Dickinson, San Jose, Calif.), magnetically activated cellsorting (MACS)(Miltenyi Biotec, Auburn, Calif.), or the like.

In other embodiments, nucleic acids are analyzed from a sample of asubset of cells. A method to separate cells, for example by using a cellsurface marker, can be employed. For example, cells can be isolated bycell sorting flow-cytometry, flow-sorting, fluorescent activated cellsorting (FACS), bead based separation such as magnetic cell sorting(MACS; e.g., using antibody coated magnetic particles), size-basedseparation (e.g., a sieve, an array of obstacles, or a filter), sortingin a microfluidics device, antibody-based separation, sedimentation,affinity adsorption, affinity extraction, or density gradientcentrifugation. Cells can be purified by laser capture microdissection.Sorting can be based on cell size, morphology, or intracellular orextracellular markers. Methods for isolating or sorting tumor cells aredescribed, for example, in Nagrath S. et al. (2007) Nature450:1235-1239; U.S. Pat. Nos. 6,008,002, 7,232,653, and 7,332,288; PCTPublication No. WO2008157220A1; and US Patent Application Nos.US20080138805A1 and US20090186065; and Rosenberg R. et al. (2002)Cytometry 49:150-158, each of which is herein incorporated by referencein their entireties.

The subset of cells can be a subset of T-cells and/or B-cells. Thesubset of T cells can be CD4+, CD8+, or CD27high cells. Cocktails ofantibodiesfor labeling and/or separating a large variety of T-cell andB-cell subsets are commercially available from vendors such as QuestDiagnostic (San Juan Capistrano, Calif.); Dako (Denmark); and the like.The following are examples of kits available for disease related subsets(where the antigen specificity of the antibodies are listed: precursorB-lymphoblastic leukemia/lymphoma (CD19, CD79a (cytoplasmic), CD20,CD10, TDt, HLADR, CD34, IgM (cytoplasmic)); diffuse large B celllymphoma (CD20, CD19, CD22, CD79a, CD30); follicular lymphoma (CD20,CD10, CD10, BCL2, BCL6); mantle cell leukemia (CD19, CD20, CD5, CD23−,BCL1); and the like.

Fluorescence-activated cell sorting (FACS) uses light scattering andfluorescent characteristics to sort cells. A fluorescent property can beimparted on a cell using, e.g., nucleic acid probes or antibodiesconjugated to a fluorescent dye. A cell suspension can form a stream offlowing liquid. The stream of cells forms drops that containapproximately one cell per drop. Before the stream forms drops, afluorescent characteristic of each cell is measured. A charge is placedon an electrical charging ring prior to fluorescence intensitymeasurement and the opposite charge is carried on the drop as it breaksfrom the stream. The charged drops pass through two high voltagedeflection plates that divert drops into different containers based upontheir charge. The charge can be directly applied to the stream and thedrop breaking off retains the charge of the same sign as the stream. Thestream is then returned to neutral after the drop breaks off.

Direct or indirect immunofluorescence can be used in FACS. In directimmunofluorescence, an antibody is directly conjugated to a fluorescentdye. In indirect immunofluorescence, the primary antibody is notlabeled, and a secondary antibody is conjugated to a fluorescent dye.

Since the identifying recombinations are present in the DNA of eachindividual's adaptive immunity cell as well as their associated RNAtranscripts, either RNA or DNA can be sequenced in the methods of theprovided invention. A recombined sequence from a T-cell or B-cellencoding a T cell receptor or immunoglobulin molecule, or a portionthereof, is referred to as a clonotype. The DNA or RNA can correspond tosequences from T-cell receptor (TCR) genes or immunoglobulin (Ig) genesthat encode antibodies. For example, the DNA and RNA can correspond tosequences encoding α, β, γ, or δ chains of a TCR. In a majority ofT-cells, the TCR is a heterodimer consisting of an α-chain and β-chain.The TCRα chain is generated by VJ recombination, and the β chainreceptor is generated by V(D)J recombination. For the TCRβ chain, inhumans there are 48 V segments, 2 D segments, and 13 J segments. Severalbases may be deleted and others added (called N and P nucleotides) ateach of the two junctions. In a minority of T-cells, the TCRs consist ofγ and δ delta chains. The TCR γ chain is generated by VJ recombination,and the TCR δ chain is generated by V(D)J recombination (Kenneth Murphy,Paul Travers, and Mark Walport, Janeway's mmunology 7th edition, GarlandScience, 2007, which is herein incorporated by reference in itsentirety).

The DNA and RNA analyzed in the methods of the invention can correspondto sequences encoding heavy chain immunoglobulins (IgH) with constantregions (α, δ, ε, γ, or μ) or light chain immunoglobulins (IgK or IgL)with constant regions λ or κ. Each antibody has two identical lightchains and two identical heavy chains. Each chain is composed of aconstant (C) and a variable region. For the heavy chain, the variableregion is composed of a variable (V), diversity (D), and joining (J)segments. Several distinct sequences coding for each type of thesesegments are present in the genome. A specific VDJ recombination eventoccurs during the development of a B-cell, marking that cell to generatea specific heavy chain. Diversity in the light chain is generated in asimilar fashion except that there is no D region so there is only VJrecombination. Somatic mutation often occurs close to the site of therecombination, causing the addition or deletion of several nucleotides,further increasing the diversity of heavy and light chains generated byB-cells. The possible diversity of the antibodies generated by a B-cellis then the product of the different heavy and light chains. Thevariable regions of the heavy and light chains contribute to form theantigen recognition (or binding) region or site. Added to this diversityis a process of somatic hypermutation which can occur after a specificresponse is mounted against some epitope.

As mentioned above, in accordance with the invention, primers may beselected to generate amplicons of subsets of recombined nucleic acidsextracted from lymphocytes. Such subsets may be referred to herein as“somatically rearranged regions.” Somatically rearranged regions maycomprise nucleic acids from developing or from fully developedlymphocytes, where developing lymphocytes are cells in whichrearrangement of immune genes has not been completed to form moleculeshaving full V(D)J regions. Exemplary incomplete somatically rearrangedregions include incomplete IgH molecules (such as, molecules containingonly D-J regions), incomplete TCRδ molecules such as, moleculescontaining only D-J regions), and inactive IgK (for example, comprisingKde-V regions). Incomplete rearrangements are also found in fullydeveloped immune cells associated with the second chromosome in a cellin which the first chromosome formed a productive rearrangement.

Controlling for Sample Amounts and Estimating Cell Numbers

Adequate sampling of the cells is an important aspect of interpretingthe repertoire data, as described further below in the definitions of“clonotype” and “repertoire.” For example, starting with 1,000 cellscreates a minimum frequency that the assay is sensitive to regardless ofhow many sequencing reads are obtained. Therefore one aspect of thisinvention is the development of methods to quantitate the number ofinput immune receptor molecules. This has been implemented for TCRβ andIgH sequences. In either case a set of primers are used that are capableof amplifying all the different sequences. In order to obtain anabsolute number of copies, a real time PCR with the multiplex of primersis performed along with a standard with a known number of immunereceptor copies. An example of real time PCR data associated with themouse vaccination example is shown in FIG. 9. This real time PCRmeasurement can be made from the amplification reaction that willsubsequently be sequenced or can be done on a separate aliquot of thesame sample. In the case of DNA, the absolute number of rearrangedimmune receptor molecules can be readily converted to number of cells(within 2 fold as some cells will have 2 rearranged copies of thespecific immune receptor assessed and others will have one). In the caseof cDNA the measured total number of rearranged molecules in the realtime sample can be extrapolated to define the total number of thesemolecules used in another amplification reaction of the same sample. Inaddition, this method can be combined with a method to determine thetotal amount of RNA to define the number of rearranged immune receptormolecules in a unit amount (say 1 μg) of RNA assuming a specificefficiency of cDNA synthesis. If the total amount of cDNA is measuredthen the efficiency of cDNA synthesis need not be considered. If thenumber of cells is also known then the rearranged immune receptor copiesper cell can be computed. If the number of cells is not known, one canestimate it from the total RNA as cells of specific type usuallygenerate comparable amount of RNA. Therefore from the copies ofrearranged immune receptor molecules per 1 μg one can estimate thenumber of these molecules per cell.

One disadvantage of doing a separate real time PCR from the reactionthat would be processed for sequencing is that there might be inhibitoryeffects that are different in the real time PCR from the other reactionas different enzymes, input DNA, and other conditions may be utilized.Processing the products of the real time PCR for sequencing wouldameliorate this problem. However low copy number using real time PCR canbe due to either low number of copies or to inhibitory effects, or othersuboptimal conditions in the reaction.

Another approach that can be utilized is to add a known amount of uniqueimmune receptor rearranged molecules with a known sequence, i.e. knownamounts of one or more internal standards, to the cDNA or genomic DNAfrom a sample of unknown quantity. By counting the relative number ofmolecules that are obtained for the known added sequence compared to therest of the sequences of the same sample, one can estimate the number ofrearranged immune receptor molecules in the initial cDNA sample. (Suchtechniques for molecular counting are well-known, e.g. Brenner et al,U.S. Pat. No. 7,537,897, which is incorporated herein by reference).Data from sequencing the added unique sequence can be used todistinguish the different possibilities if a real time PCR calibrationis being used as well. Low copy number of rearranged immune receptor inthe DNA (or cDNA) would create a high ratio between the number ofmolecules for the spiked sequence compared to the rest of the samplesequences. On the other hand, if the measured low copy number by realtime PCR is due to inefficiency in the reaction, the ratio would not behigh.

In one aspect, the invention provides methods for measuring clonotypeexpression at a cellular level. That is, as noted above, clonotypes maybe used to count lymphocytes; therefore, by measuring clonotypes derivedfrom genomic DNA and the same clonotypes derived from RNA, cell-basedexpression of clonotypes may be determined A method for simultaneouslymeasuring lymphocyte numbers and clonotype expression levels in a samplemay comprise the steps of: (a) obtaining from an individual a samplecomprising T cells and/or B cells; (b) sequencing spatially isolatedindividual molecules derived from genomic DNA of said cells, suchspatially isolated individual molecules comprising a number ofclonotypes corresponding to a number of lymphocytes in the sample; (c)sequencing spatially isolated individual molecules derived from RNA ofsaid cells, such spatially isolated individual molecules comprisingnumbers of clonotypes corresponding to expression levels thereof in thelymphocytes of the sample; and (d) determining clonotype expressionlevels in lymphocytes of the sample by comparing for each clonotype thenumber determined from isolated individual molecules derived fromgenomic DNA of said cells and the number determined from isolatedindividual molecules derived from RNA of said cells. Genomic DNA and RNAare readily extracted from the same sample using commercially availablekits, such as the AllPrep DNA/RNA Mini Kit (Qiagen GmbH, Germany). Asmentioned above, in one embodiment, the step of determining furtherincludes determining said number of lymphocytes in said sample by addinga known quantity of an internal standard to said genomic DNA. In anotherembodiment, where for example the sample is peripheral blood, the samplehas a defined volume which permits a concentration of said lymphocytesto be determined in said sample. Typically, such a defined volume is inthe range of from 1 mL to 50 mL, and more usually, in the range of from1 mL to 10 mL. In another embodiment, numbers of the same clonotypederived from genomic DNA and RNA are compared by simply dividing thenumber of clonotypes determined from the isolated individual moleculesderived from the RNA by the number of clonotypes determined from theisolated individual molecules derived from said genomic DNA. Such twosets of clonotypes are readily distinguished in the same sequencing runby the use of labels, particularly oligonucleotide tags that areattached during the sample preparation process. For Solexa-basedsequencing, such labels may be incorporated with the tags used toidentify different samples by (for example) adding a single nucleotideto the tag to indicate DNA or RNA, or simply using an additional tag sothat each patient sample is labeled with two tags, one for the genomicDNA fraction and one for the RNA fraction. Thus, said step of sequencingsaid spatially isolation individual molecules derived from said RNA mayinclude labeling each of said spatially isolated individual moleculeswith a first label indicating its RNA origin and said step of sequencingsaid spatially isolation individual molecules derived from said genomicDNA may include labeling each of said spatially isolated individualmolecules with a second label indicating its genomic DNA origin suchthat the first label is distinguishable from the second label. In oneembodiment, such labels are distinct oligonucleotide tags that areidentified by sequencing.

Likewise, the invention may be used to provide simultaneously (that is,based on measurements on a single sample) lymphocyte number andclonality. Such embodiment may be implemented with the following steps:(a) obtaining from an individual a sample comprising T cells and/or Bcells; (b) sequencing spatially isolated individual molecules derivedfrom nucleic acid of said cells, such spatially isolated individualmolecules comprising a number of clonotypes corresponding to a number oflymphocytes in the sample; (c) determining the number of lymphocytesfrom the number of spatially isolated individual molecules; (d)determining abundances of different sequences of the spatially isolatedindividual molecules to generate a clonotype profile and a measure ofclonality based thereon. The nucleic acid from the lymphocytes may begenomic DNA and/or RNA; however, preferably the nucleic acid is genomicDNA Similarly as above, in one embodiment, the step of determining saidnumber further includes determining said number of lymphocytes in saidsample by adding a known quantity of an internal standard to saidgenomic DNA. And similarly, when the sample is a peripheral blood sampleit has a defined volume so that a concentration of said lymphocytes insaid sample is determined. In some embodiments of the above, only Bcells are employed and in other embodiments only T cells are employed.

Amplification of Nucleic Acid Populations

As noted below, amplicons of target populations of nucleic acids may begenerated by a variety of amplification techniques. In one aspect of theinvention, multiplex PCR is used to amplify members of a mixture ofnucleic acids, particularly mixtures comprising recombined immunemolecules such as T cell receptors, B cell receptors, or portionsthereof. Guidance for carrying out multiplex PCRs of such immunemolecules is found in the following references, which are incorporatedby reference: Morley, U.S. Pat. No. 5,296,351; Gorski, U.S. Pat. No.5,837,447; Dau, U.S. Pat. No. 6,087,096; Von Dongen et al, U.S. patentpublication 2006/0234234; European patent publication EP 1544308B1; andthe like. The foregoing references describe the technique referred to as“spectratyping,” where a population of immune molecules are amplified bymultiplex PCR after which the sequences of the resulting amplicon arephysically separated, e.g. by electrophoresis, in order to determinewhether there is a predominant size class. Such a class would indicate apredominant clonal population of lymphocytes which, in turn, would beindicative of disease state. In spectratyping, it is important to selectprimers that display little or no cross-reactivity (i.e. that do notanneal to binding sites of other primers); otherwise there may be afalse representation of size classes in the amplicon. In the presentinvention, so long as the nucleic acids of a population are uniformlyamplified, cross-reactivity of primers is permissible because thesequences of the amplified nucleic acids are analyzed in the presentinvention, not merely their sizes. As described more fully below, in oneaspect, the step of spatially isolating individual nucleic acidmolecules is achieved by carrying out a primary multiplex amplificationof a preselected somatically rearranged region or portion thereof (i.e.target sequences) using forward and reverse primers that each have tailsnon-complementary to the target sequences to produce a first ampliconwhose member sequences have common sequences at each end that allowfurther manipulation. For example, such common ends may include primerbinding sites for continued amplification using just a single forwardprimer and a single reverse primer instead of multiples of each, or forbridge amplification of individual molecules on a solid surface, or thelike. Such common ends may be added in a single amplification asdescribed above, or they may be added in a two-step procedure to avoiddifficulties associated with manufacturing and exercising qualitycontrol over mixtures of long primers (e.g. 50-70 bases or more). Insuch a two-step process (described more fully below and illustrated inFIGS. 4A-4B), the primary amplification is carried out as describedabove, except that the primer tails are limited in length to provideonly forward and reverse primer binding sites at the ends of thesequences of the first amplicon. A secondary amplification is thencarried out using secondary amplification primers specific to theseprimer binding sites to add further sequences to the ends of a secondamplicon. The secondary amplification primers have tailsnon-complementary to the target sequences, which form the ends of thesecond amplicon and which may be used in connection with sequencing theclonotypes of the second amplicon. In one embodiment, such addedsequences may include primer binding sites for generating sequence readsand primer binding sites for carrying out bridge PCR on a solid surfaceto generate clonal populations of spatially isolated individualmolecules, for example, when Solexa-based sequencing is used. In thislatter approach, a sample of sequences from the second amplicon aredisposed on a solid surface that has attached complementaryoligonucleotides capable of annealing to sequences of the sample, afterwhich cycles of primer extension, denaturation, annealing areimplemented until clonal populations of templates are formed.Preferably, the size of the sample is selected so that (i) it includesan effective representation of clonotypes in the original sample, and(ii) the density of clonal populations on the solid surface is in arange that permits unambiguous sequence determination of clonotypes.

In addition to ensuring that the sample contains sufficient cells to berepresentative of the original sample, it is important that theamplicons generated by the multiplex PCR reaction be representative ofthe cells in the reaction. In order to achieve this, primer conditionsshould be selected such that amplification from every cell in thereaction occurs.

TCR or BCR sequences or portions thereof can be amplified from nucleicacid in a multiplex reaction using at least one primer that anneals tothe C region and one or more primers that can anneal to one or more Vsegments (as illustrated in FIGS. 2A-2B and FIGS. 4A-4B and discussedmore fully below). The number of primers that anneal to V segments in amultiplex reaction can be, for example, at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or80. The number of primers that anneal to V segments in a multiplexreaction can be, for example, 10-60, 20-50, 30-50, 40-50, 20-40, 30-40,or 35-40. The primers can anneal to different V segments. For IgH genes,because of the possibility of somatic mutations in the V segments,multiple primers that anneal to each V segment can be used; for example,1, 2, 3, 4, or 5 primers per V segment. The number of primers thatanneal to C segments in a multiplex reaction can include, for example,at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Thenumber of primers that anneal to C segments in a multiplex reaction canbe 1-10, 2-9, 3-8, 4-7, 3-8, or 3-6. Amplification of TCR orimmunoglobulin genes can occur as described in Example 3 and/or Example4.

The region to be amplified can include the full clonal sequence or asubset of the clonal sequence, including the V-D junction, D-J junctionof an immunoglobulin or T-cell receptor gene, the full variable regionof an immunoglobulin or T-cell receptor gene, the antigen recognitionregion, or a CDR, e.g., complementarity determining region 3 (CDR3).

The TCR or immunoglobulin sequence can amplified using a primary and asecondary amplification step. Each of the different amplification stepscan comprise different primers. The different primers can introducesequence not originally present in the immune gene sequence. Forexample, the amplification procedure can add new primer binding sites tothe ends of the target sequences to convert a multiplex amplification toa singleplex amplification or the amplification procedure can add one ormore tags to the 5′ and/or 3′ end of amplified TCR or immunoglobulinsequence as illustrated in FIGS. 3A-3C). The tag can be sequence thatfacilitates subsequent sequencing of the amplified DNA. The tag can besequence that facilitates binding the amplified sequence to a solidsupport.

Other methods for amplification may not employ any primers in the Vregion. Instead, a specific primer can be used from the C segment and ageneric primer can be put in the other side 5′). The generic primer canbe appended in the cDNA synthesis through different methods includingthe well described methods of strand switching. Similarly, the genericprimer can be appended after cDNA making through different methodsincluding ligation.

Other means of amplifying nucleic acid that can be used in the methodsof the provided invention include, for example, reversetranscription-PCR, real-time PCR, quantitative real-time PCR, digitalPCR (dPCR), digital emulsion PCR (dePCR), clonal PCR, amplified fragmentlength polymorphism PCR (AFLP PCR), allele specific PCR, assembly PCR,asymmetric PCR (in which a great excess of primers for a chosen strandis used), colony PCR, helicase-dependent amplification (HDA), Hot StartPCR, inverse PCR (IPCR), in situ PCR, long PCR (extension of DNA greaterthan about 5 kilobases), multiplex PCR, nested PCR (uses more than onepair of primers), single-cell PCR, touchdown PCR, loop-mediatedisothermal PCR (LAMP), and nucleic acid sequence based amplification(NASBA). Other amplification schemes include: Ligase Chain Reaction,Branch DNA Amplification, Rolling Circle Amplification, Circle to CircleAmplification, SPIA amplification, Target Amplification by Capture andLigation (TACL) amplification, and RACE amplification.

The information in RNA in a sample can be converted to cDNA by usingreverse transcription. PolyA primers, random primers, and/or genespecific primers can be used in reverse transcription reactions inaccordance with conventional protocols.

After amplification of DNA from the genome (or amplification of nucleicacid in the form of cDNA by reverse transcribing RNA), the individualnucleic acid molecules can be isolated, optionally re-amplified, andthen sequenced individually. Exemplary amplification protocols may befound in van Dongen et al, Leukemia, 17: 2257-2317 (2003) or van Dongenet al, U.S. patent publication 2006/0234234, which is incorporated byreference. Briefly, an exemplary protocol is as follows: Reactionbuffer: ABI Buffer II or ABI Gold Buffer Life Technologies, San Diego,Calif.); 50 μL final reaction volume; 100 ng sample DNA; 10 pmol of eachprimer subject to adjustments to balance amplification as describedbelow); dNTPs at 200 μM final concentration; MgCl₂ at 1.5 mM finalconcentration (subject to optimization depending on target sequences andpolymerase); Taq polymerase (1-2 U/tube); cycling conditions:preactivation 7 min at 95° C.; annealing at 60° C.; cycling times: 30 sdenaturation; 30 s annealing; 30 s extension.

Polymerases that can be used for amplification in the methods of theinvention are commercially available and include, for example, Taqpolymerase, AccuPrime polymerase, or Pfu. The choice of polymerase touse can be based on whether fidelity or efficiency is preferred.

Methods for isolation of nucleic acids from a pool include spatialseparation of the molecules in two dimensions on a solid substrate(e.g., glass slide), spatial separation of the molecules in threedimensions in a solution within micelles (such as can be achieved usingoil emulsions with or without immobilizing the molecules on a solidsurface such as beads), or using microreaction chambers in, for example,microfluidic or nano-fluidic chips. Dilution can be used to ensure thaton average a single molecule is present in a given volume, spatialregion, bead, or reaction chamber. Guidance for such methods ofisolating individual nucleic acid molecules is found in the followingreferences: Sambrook, Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 2001s); Shendure et al, Science, 309:1728-1732 (including supplemental material)(2005); U.S. Pat. No.6,300,070; Bentley et al, Nature, 456: 53-59 (including supplementalmaterial)(2008); U.S. Pat. No. 7,323,305; Matsubara et al, Biosensors &Bioelectronics, 20: 1482-1490 (2005): U.S. Pat. No. 6,753,147; and thelike.

Real time PCR, picogreen staining, nanofluidic electrophoresis (e.g.LabChip) or UV absorption measurements can be used in an initial step tojudge the functional amount of amplifiable material.

Methods for re-amplification of nucleic acids include bacterial growthof isolated colonies transformed with nucleic acid, amplification on aslide (e.g., PCR colonies (polonies)), and amplification on a bead (e.g.in an emulsion PCR). The same method can be used to amplify andre-amplify the nucleic acid or a different method can be used to amplifyand reamplify the nucleic acid.

In certain embodiments the subcloning steps include a step in which acommon primer is attached to the DNA or RNA through an amplification orligation step. This primer is then used to amplify the clones and as arecognition sequence for hybridization of a primer for sequencing (e.g.as illustrated in FIGS. 2A-2B and 4A-4B, and discussed more fullybelow).

In one aspect, multiplex amplifications are carried out so that relativeamounts of sequences in a starting population are substantially the sameas those in the amplified population, or amplicon. That is, multiplexamplifications are carried out with minimal amplification bias amongmember sequences of a sample population. In one embodiment, suchrelative amounts are substantially the same if each relative amount inan amplicon is within five fold of its value in the starting sample. Inanother embodiment, such relative amounts are substantially the same ifeach relative amount in an amplicon is within two fold of its value inthe starting sample. As discussed more fully below, amplification biasin PCR may be detected and corrected using conventional techniques sothat a set of PCR primers may be selected for a predetermined repertoirethat provide unbiased amplification of any sample.

In regard to many repertoires based on TCR or BCR sequences, a multiplexamplification optionally uses all the V segments. The reaction isoptimized to attempt to get amplification that maintains the relativeabundance of the sequences amplified by different V segment primers.Some of the primers are related, and hence many of the primers may“cross talk,” amplifying templates that are not perfectly matched withit. The conditions are optimized so that each template can be amplifiedin a similar fashion irrespective of which primer amplified it. In otherwords if there are two templates, then after 1,000 fold amplificationboth templates can be amplified approximately 1,000 fold, and it doesnot matter that for one of the templates half of the amplified productscarried a different primer because of the cross talk. In subsequentanalysis of the sequencing data the primer sequence is eliminated fromthe analysis, and hence it does not matter what primer is used in theamplification as long as the templates are amplified equally.

Since the amount of each template is not known in a cDNA populationgenerated from mRNA, a set of standards may be generated usingsingleplex PCRs of a cDNA population of clonotypes. This was carried outfor a repertoire of TCRβ clonotypes. The product in each of 34 such PCRs(using in the separate reactions primers of Example 3) comprised aplurality of sequences with one V primer. The different products werecarefully quantitated to create a set of standards at the sameconcentration. A pool of all 34 primers was used and 34 real time PCRswere performed using the pool of primers and each of the standardsequences as a template. Ideally without bias all the 34 standards willshow equal efficiency of amplification by real time PCR. That suggeststhat each sequence is amplified equally even though the presence ofcross talk makes it unclear what primers are carrying out theamplification. This optimization is consistent with the goal of havingequal amplification irrespective of the actual primers that isincorporated in the amplification product. Increasing the total primerpool concentration significantly reduced the dynamic range as expectedfrom increasing the efficiency of the amplification. Furthermore fortemplates that seemed to amplify more efficiently than the average, theconcentration of their perfectly matched primer in the pool wasdecreased. Conversely for templates that were inefficiently amplifiedthe concentration of their perfectly matched primer was increased. Thisoptimization demonstrated that all the templates are amplified withintwo fold of the average amplification.

Amplification bias may also be avoided by carrying out a two-stageamplification (as illustrated in FIGS. 2A-2B) wherein a small number ofamplification cycles are implemented in a first, or primary, stage usingprimers having tails non-complementary with the target sequences. Thetails include primer binding sites that are added to the ends of thesequences of the primary amplicon so that such sites are used in asecond stage amplification using only a single forward primer and asingle reverse primer, thereby eliminating a primary cause ofamplification bias. Preferably, the primary PCR will have a small enoughnumber of cycles (e.g. 5-10) to minimize the differential amplificationby the different primers. The secondary amplification is done with onepair of primers and hence the issue of differential amplification isminimal. One percent of the primary PCR is taken directly to thesecondary PCR. Thirty-five cycles (equivalent to −28 cycles without the100 fold dilution step) used between the two amplifications weresufficient to show a robust amplification irrespective of whether thebreakdown of cycles were: one cycle primary and 34 secondary or 25primary and 10 secondary. Even though ideally doing only 1 cycle in theprimary PCR may decrease the amplification bias, there are otherconsiderations. One aspect of this is representation. This plays a rolewhen the starting input amount is not in excess to the number of readsultimately obtained. For example, if 1,000,000 reads are obtained andstarting with 1,000,000 input molecules then taking only representationfrom 100,000 molecules to the secondary amplification would degrade theprecision of estimating the relative abundance of the different speciesin the original sample. The 100 fold dilution between the 2 steps meansthat the representation is reduced unless the primary PCR amplificationgenerated significantly more than 100 molecules. This indicates that aminimum 8 cycles (256 fold), but more comfortably 10 cycle (1,000 fold),may be used. The alternative to that is to take more than 1% of theprimary PCR into the secondary but because of the high concentration ofprimer used in the primary PCR, a big dilution factor is can be used toensure these primers do not interfere in the amplification and worsenthe amplification bias between sequences. Another alternative is to adda purification or enzymatic step to eliminate the primers from theprimary PCR to allow a smaller dilution of it. In this example, theprimary PCR was 10 cycles and the second 25 cycles.

The reproducibility of a multiplex PCR may be assessed as follows, asexemplified with the test set of primers from Example 2. Two primary PCRreactions are performed using the test set of primers, e.g. the pooledTCRβ primers and the C primer (of Example 2) and one cDNA sample as atemplate. The relative abundance in each amplified template is assessedusing real time PCR. Using each of the two amplified products as atemplate, thirty four different real time PCR reactions were performedusing the C primer and one of the V primers in each reaction. The datashown in FIG. 5 demonstrate that the relative abundance determined byreal time PCR was highly reproducible using all the V primers for twosamples, indicating that the multiplexed amplification is highlyreproducible. The cycle number (Ct value) for each of the real time PCRamplifications using the one multiplexed amplification product as atemplate is shown on the X axis and using the second multiplexedamplification product as a template is depicted on the Y axis.

The amount of amplification bias of a set of primers may be assessedusing the following procedure, which is exemplified using the primer setof Example 2. The test primer set (as above) is used to amplify a cDNAas a template (e.g. obtained from mRNA extracted from lymphocytes). Theamount of template amplified by each of the 34 different primers (alongwith C segment primer) is determined using real time PCR and that amountis compared with the amount amplified using the same primers with thecDNA. Since there may be cross talk even if the relative abundance amongthe internal sequences in the amplified product and the cDNA were thesame, only significant differences in amplification may be detectedusing this readout. This possibility may be tested by synthesizingcollection of primers to amplify internal segments of a number of thestarting cDNA sequences. For example, 12 oligos were designed that can,when used with C segment primer, amplify sequences internal to the aboveV segment primers. If there is minimal amplification bias, then theconcentration of these internal sequences should change little betweenthe starting cDNA and the amplified products. Data from this example isshown in FIG. 6. There, a cDNA sample was used as a template for amultiplexed amplification using the pooled TCRβ primers and the C primer(from Example 2). The C primer and the downstream internal primers wereused for the initial amplification of template material from themultiplex amplification. Similarly real time PCR was used to assess therelative abundance of these same sequences in the cDNA. If themultiplexed amplification had any significant biases, the relativeabundance in the amplified material would be very different from that inthe cDNA. As can be seen in FIG. 6, high correlation was seendemonstrating minimal amplification bias in the multiplexedamplification. The cycle number (Ct value) for each of the real time PCRamplification using internal primers, and cDNA and the multiplexedamplification product as template is shown on X and Y axis,respectively.

The initial amplification can be done from DNA or RNA (e.g., afterconversion to cDNA).

Sequencing Nucleic Acid Populations

Any high-throughput technique for sequencing nucleic acids can be usedin the method of the invention. DNA sequencing techniques includedideoxy sequencing reactions (Sanger method) using labeled terminatorsor primers and gel separation in slab or capillary, sequencing bysynthesis using reversibly terminated labeled nucleotides,pyrosequencing, 454 sequencing, allele specific hybridization to alibrary of labeled oligonucleotide probes, sequencing by synthesis usingallele specific hybridization to a library of labeled clones that isfollowed by ligation, real time monitoring of the incorporation oflabeled nucleotides during a polymerization step, polony sequencing, andSOLiD sequencing. Sequencing of the separated molecules has morerecently been demonstrated by sequential or single extension reactionsusing polymerases or ligases as well as by single or sequentialdifferential hybridizations with libraries of probes. These reactionshave been performed on many clonal sequences in parallel includingdemonstrations in current commercial applications of over 100 millionsequences in parallel. These sequencing approaches can thus be used tostudy the repertoire of T-cell receptor (TCR) and/or B-cell receptor(BCR). In one aspect of the invention, high-throughput methods ofsequencing are employed that comprise a step of spatially isolatingindividual molecules on a solid surface where they are sequenced inparallel. Such solid surfaces may include nonporous surfaces (such as inSolexa sequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) orComplete Genomics sequencing, e.g. Drmanac et al, Science, 327: 78-81(2010)), arrays of wells, which may include bead- or particle-boundtemplates (such as with 454, e.g. Margulies et al, Nature, 437: 376-380(2005) or Ion Torrent sequencing, U.S. patent publication 2010/0137143or 2010/0304982), micromachined membranes (such as with SMRT sequencing,e.g. Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as withSOLiD sequencing or polony sequencing, e.g. Kim et al, Science, 316:1481-1414 (2007)). In another aspect, such methods comprise amplifyingthe isolated molecules either before or after they are spatiallyisolated on a solid surface. Prior amplification may compriseemulsion-based amplification, such as emulsion PCR, or rolling circleamplification. Of particular interest is Solexa-based sequencing whereindividual template molecules are spatially isolated on a solid surface,after which they are amplified in parallel by bridge PCR to formseparate clonal populations, or clusters, and then sequenced, asdescribed in Bentley et al (cited above) and in manufacturer'sinstructions (e.g. TruSeq™ Sample Preparation Kit and Data Sheet,Illumina, Inc., San Diego, Calif., 2010); and further in the followingreferences: U.S. Pat. Nos. 6,090,592; 6,300,070; 7,115,400; andEP0972081B1; which are incorporated by reference. In one embodiment,individual molecules disposed and amplified on a solid surface formclusters in a density of at least 10⁵ clusters per cm²; or in a densityof at least 5×10⁵ per cm²; or in a density of at least 10⁶ clusters percm². In one embodiment, sequencing chemistries are employed havingrelatively high error rates. In such embodiments, the average qualityscores produced by such chemistries are monotonically decliningfunctions of sequence read lengths. In one embodiment, such declinecorresponds to 0.5 percent of sequence reads have at least one error inpositions 1-75; 1 percent of sequence reads have at least one error inpositions 76-100; and 2 percent of sequence reads have at least oneerror in positions 101-125.

In one aspect, for each sample from an individual, the sequencingtechnique used in the methods of the invention generates sequences ofleast 1000 clonotypes per run; in another aspect, such techniquegenerates sequences of at least 10,000 clonotypes per run; in anotheraspect, such technique generates sequences of at least 100,000clonotypes per run; in another aspect, such technique generatessequences of at least 500,000 clonotypes per run; and in another aspect,such technique generates sequences of at least 1,000,000 clonotypes perrun. In still another aspect, such technique generates sequences ofbetween 100,000 to 1,000,000 clonotypes per run per individual sample.

The sequencing technique used in the methods of the provided inventioncan generate about 30 bp, about 40 bp, about 50 bp, about 60 bp, about70 bp, about 80 bp, about 90 bp, about 100 bp, about 110, about 120 bpper read, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about350 bp, about 400 bp, about 450 bp, about 500 bp, about 550 bp, or about600 bp per read.

The sequencing technique used in the methods of the provided inventioncan generate at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150,200, 250, 300, 350, 400, 450, 500, 550, or 600 bp per read. In oneaspect, a sequence-based clonotype profile of an individual is obtainedusing the following steps: (a) obtaining a nucleic acid sample fromT-cells and/or B-cells of the individual; (b) spatially isolatingindividual molecules derived from such nucleic acid sample, theindividual molecules comprising nested sets of templates each generatedfrom a nucleic acid in the sample and each containing a somaticallyrearranged region or a portion thereof, each nested set being capable ofproducing a plurality of sequence reads each extending in the samedirection and each starting from a different position on the nucleicacid from which the nested set was generated; (c) sequencing saidspatially isolated individual molecules; and (d) determining abundancesof different sequences of the nucleic acid molecules from the nucleicacid sample to generate the clonotype profile. In one embodiment, thestep of sequencing includes producing a plurality of sequence reads foreach of the nested sets. In another embodiment, each of the somaticallyrearranged regions comprise a V region and a J region, and each of theplurality of sequence reads starts from a different position in the Vregion and extends in the direction of its associated J region. Inanother embodiment, the step of sequencing comprises bidirectionallysequencing each of the spatially isolated individual molecules toproduce at least one forward sequence read and at least one reversesequence read. Further to the latter embodiment, at least one of theforward sequence reads and at least one of the reverse sequence readshave an overlap region such that bases of such overlap region aredetermined by a reverse complementary relationship between such sequencereads. In still another embodiment, each of the somatically rearrangedregions comprise a V region and a J region and the step of sequencingfurther includes determining a sequence of each of the individualnucleic acid molecules from one or more of its forward sequence readsand at least one reverse sequence read starting from a position in a Jregion and extending in the direction of its associated V region. Inanother embodiment, individual molecules comprise nucleic acids selectedfrom the group consisting of complete IgH molecules, incomplete IgHmolecules, complete IgK complete, IgK inactive molecules, TCRβmolecules, TCRγ molecules, complete TCRδ molecules, and incomplete TCRδmolecules. In another embodiment, the step of sequencing comprisesgenerating the sequence reads having monotonically decreasing qualityscores. Further to the latter embodiment, monotonically decreasingquality scores are such that the sequence reads have error rates nobetter than the following: 0.2 percent of sequence reads contain atleast one error in base positions 1 to 50, 0.2 to 1.0 percent ofsequence reads contain at least one error in positions 51-75, 0.5 to 1.5percent of sequence reads contain at least one error in positions76-100.

As noted below in the definition of repertoire, different predeterminedregions of immunoglobulin or T cell receptor genes can be sequenced. Insome embodiments, the full sequence of the variable regions can besequenced to identify and quantify a clonotype.

A unique subset of the full clonal sequences can be sequenced. In someembodiments, nucleotides comprising the VD and the DJ junctions aresequenced to uniquely identify and quantify a clonotype. In otherembodiments, the fragment that can be sequenced is the full variableregion. In yet another embodiment, the antigen recognition region or thecomplementarity determining region 3 (CDR3) is sequenced. A fragmentcontaining the full CDR3 or the full variable region can be amplified toallow the sequencing of the CDR3 comprising parts of the V, D, and Jsegments.

In one embodiment, only the CDR3 is amplified and sequenced.Amplification and sequencing of the CDR3 can be accomplished by usingprimers specific to one or more V segment sequences (as well as one ormore primer(s) on the other side of the amplicon in the C segment).Primers for each of the V segments can be utilized in one or moreamplification reactions leading to the amplification of the fullrepertoire of sequences. This repertoire of sequences can then be mixedand subjected to separation, with or without amplification, andsequenced using any of the sequencing techniques described. When theamplification with the various V primers is done in separate tubes, thenumber of molecules carrying the different V segments can be“normalized” due to PCR saturation. For example, if one particular Vsegment had one or several clonal expansions leading to itsrepresentation more than other segments this information may be erasedor decreased since the PCR reaction for each segment can be driven tosaturation or close to it. Real time PCR can be used to quantify howmuch of each V segment is present. The full CDR3 can be sequenced, or asubset of the sequence CDR3 can be sequenced.

In one embodiment, only a subset of clonotypes is analyzed. This can beaccomplished by amplifying with a primer specific to the subset ofclonotypes, for example, a primer that is specific to the V segment.Unique clonotypes can be identified by sequencing with long contiguousreads that provide full connectivity. In some embodiments, when severalsequences of interest are present, a short read length across only oneof the junctions can generate degenerate tags that are not unique to aspecific clonotype but are shared among multiple clonotypes. For examplesequencing across the V/J junction can lump all the sequences with thesame V/J irrespective of the D segment as one clonotype. Information onthe full connectivity of all segments allows sequences to bedistinguished that may share the same V and J segments but are connectedto different D segments, for example.

Clonotype Determination from Sequence Data

In one aspect of the invention, sequences of clonotypes (including butnot limited to those derived from IgH, TCRα, TCRβ, TCRγ, TCRδ, and/orIgLκ IgK)) may be determined by combining information from one or moresequence reads, for example, along the V(D)J regions of the selectedchains. In another aspect, sequences of clonotypes are determined bycombining information from a plurality of sequence reads. (As usedherein, a “sequence read” is a sequence of data generated by asequencing technique from which a sequence of nucleotides is determined.Typically, sequence reads are made by extending a primer along atemplate nucleic acid, e.g. with a DNA polymerase or a DNA ligase. Datais generated by recording signals, such as optical, chemical (e.g. pHchange), or electrical signals, associated with such extension.) Suchpluralities of sequence reads may include one or more sequence readsalong a sense strand i.e. “forward” sequence reads) and one or moresequence reads along its complementary strand i.e. “reverse” sequencereads). When multiple sequence reads are generated along the samestrand, separate templates are first generated by amplifying samplemolecules with primers selected for the different positions of thesequence reads. This concept is illustrated in FIG. 4A where primers(404, 406 and 408) are employed to generate amplicons (410, 412, and414, respectively) in a single reaction. Such amplifications may becarried out in the same reaction or in separate reactions. In oneaspect, whenever PCR is employed, separate amplification reactions areused for generating the separate templates which, in turn, are combinedand used to generate multiple sequence reads along the same strand. Thislatter approach is preferable for avoiding the need to balance primerconcentrations (and/or other reaction parameters) to ensure equalamplification of the multiple templates sometimes referred to herein as“balanced amplification” or “unbiased amplification”). The generation oftemplates in separate reactions is illustrated in FIGS. 4B-4C. There asample containing IgH (400) is divided into three portions (472, 474,and 476) which are added to separate PCRs using J region primers (401)and V region primers (404, 406, and 408, respectively) to produceamplicons (420, 422 and 424, respectively). The latter amplicons arethen combined (478) in secondary PCR (480) using P5 and P7 primers toprepare the templates (482) for bridge PCR and sequencing on an IlluminaGA sequencer, or like instrument.

Sequence reads of the invention may have a wide variety of lengths,depending in part on the sequencing technique being employed. Forexample, for some techniques, several trade-offs may arise in itsimplementation, for example, (i) the number and lengths of sequencereads per template and (ii) the cost and duration of a sequencingoperation. In one embodiment, sequence reads are in the range of from 20to 400 nucleotides; in another embodiment, sequence reads are in a rangeof from 30 to 200 nucleotides; in still another embodiment, sequencereads are in the range of from 30 to 120 nucleotides. In one embodiment,1 to 4 sequence reads are generated for determining the sequence of eachclonotype; in another embodiment, 2 to 4 sequence reads are generatedfor determining the sequence of each clonotype; and in anotherembodiment, 2 to 3 sequence reads are generated for determining thesequence of each clonotype. In the foregoing embodiments, the numbersgiven are exclusive of sequence reads used to identify samples fromdifferent individuals. The lengths of the various sequence reads used inthe embodiments described below may also vary based on the informationthat is sought to be captured by the read; for example, the startinglocation and length of a sequence read may be designed to provide thelength of an NDN region as well as its nucleotide sequence; thus,sequence reads spanning the entire NDN region are selected. In otheraspects, one or more sequence reads encompasses the D and/or NDNregions.

In another aspect of the invention, sequences of clonotypes aredetermined in part by aligning sequence reads to one or more V regionreference sequences and one or more J region reference sequences, and inpart by base determination without alignment to reference sequences,such as in the highly variable NDN region. A variety of alignmentalgorithms may be applied to the sequence reads and reference sequences.For example, guidance for selecting alignment methods is available inBatzoglou, Briefings in Bioinformatics, 6: 6-22 (2005), which isincorporated by reference. In one aspect, whenever V reads or C reads(described more fully below) are aligned to V and J region referencesequences, a tree search algorithm is employed, e.g. Cormen et al,Introduction to Algorithms, Third Edition (The MIT Press, 2009). Thecodon structures of V and J reference sequences may be used in analignment process to remove sequencing errors and/or to determine aconfidence level in the resulting alignment, as described more fullybelow. In another aspect, an end of at least one forward read and an endof at least one reverse read overlap in an overlap region (e.g. 308 inFIG. 3B), so that the bases of the reads are in a reverse complementaryrelationship with one another. Thus, for example, if a forward read inthe overlap region is “5′-acgttgc”, then a reverse read in a reversecomplementary relationship is “5′-gcaacgt” within the same overlapregion. In one aspect, bases within such an overlap region aredetermined, at least in part, from such a reverse complementaryrelationship. That is, a likelihood of a base call (or a related qualityscore) in a prospective overlap region is increased if it preserves, oris consistent with, a reverse complementary relationship between the twosequence reads. In one aspect, clonotypes of TCR β and IgH chainsillustrated in FIG. 3B) are determined by at least one sequence readstarting in its J region and extending in the direction of itsassociated V region referred to herein as a “C read” 304)) and at leastone sequence read starting in its V region and extending in thedirection of its associated J region referred to herein as a “V read”(306)). Overlap region (308) may or may not encompass the NDN region(315) as shown in FIG. 3B. Overlap region (308) may be entirely in the Jregion, entirely in the NDN region, entirely in the V region, or it mayencompass a J region-NDN region boundary or a V region-NDN regionboundary, or both such boundaries (as illustrated in FIG. 3B).Typically, such sequence reads are generated by extending sequencingprimers, e.g. (302) and (310) in FIG. 3B, with a polymerase in asequencing-by-synthesis reaction, e.g. Metzger, Nature Reviews Genetics,11: 31-46 (2010); Fuller et al, Nature Biotechnology, 27: 1013-1023(2009). The binding sites for primers (302) and (310) are predetermined,so that they can provide a starting point or anchoring point for initialalignment and analysis of the sequence reads. In one embodiment, a Cread is positioned so that it encompasses the D and/or NDN region of theTCR β or IgH chain and includes a portion of the adjacent V region, e.g.as illustrated in FIGS. 3B and 3C. In one aspect, the overlap of the Vread and the C read in the V region is used to align the reads with oneanother. In other embodiments, such alignment of sequence reads is notnecessary, e.g. with TCRβ chains, so that a V read may only be longenough to identify the particular V region of a clonotype. This latteraspect is illustrated in FIG. 3C. Sequence read (330) is used toidentify a V region, with or without overlapping another sequence read,and another sequence read (332) traverses the NDN region and is used todetermine the sequence thereof. Portion (334) of sequence read (332)that extends into the V region is used to associate the sequenceinformation of sequence read (332) with that of sequence read (330) todetermine a clonotype. For some sequencing methods, such as base-by-baseapproaches like the Solexa sequencing method, sequencing run time andreagent costs are reduced by minimizing the number of sequencing cyclesin an analysis. Optionally, as illustrated in FIG. 3B, amplicon (300) isproduced with sample tag (312) to distinguish between clonotypesoriginating from different biological samples, e.g. different patients.Sample tag (312) may be identified by annealing a primer to primerbinding region (316) and extending it (314) to produce a sequence readacross tag (312), from which sample tag (312) is decoded.

The IgH chain is more challenging to analyze than TCRβ chain because ofat least two factors: i) the presence of somatic mutations makes themapping or alignment more difficult, and ii) the NDN region is larger sothat it is often not possible to map a portion of the V segment to the Cread. In one aspect of the invention, this problem is overcome by usinga plurality of primer sets for generating V reads, which are located atdifferent locations along the V region, preferably so that the primerbinding sites are nonoverlapping and spaced apart, and with at least oneprimer binding site adjacent to the NDN region, e.g. in one embodimentfrom 5 to 50 bases from the V-NDN junction, or in another embodimentfrom 10 to 50 bases from the V-NDN junction. The redundancy of aplurality of primer sets minimizes the risk of failing to detect aclonotype due to a failure of one or two primers having binding sitesaffected by somatic mutations. In addition, the presence of at least oneprimer binding site adjacent to the NDN region makes it more likely thata V read will overlap with the C read and hence effectively extend thelength of the C read. This allows for the generation of a continuoussequence that spans all sizes of NDN regions and that can also mapsubstantially the entire V and J regions on both sides of the NDNregion. Embodiments for carrying out such a scheme are illustrated inFIGS. 4A and 4D. In FIG. 4A, a sample comprising IgH chains (400) aresequenced by generating a plurality amplicons for each chain byamplifying the chains with a single set of J region primers (401) and aplurality (three shown) of sets of V region (402) primers (404, 406,408) to produce a plurality of nested amplicons (e.g., 410, 412, 416)all comprising the same NDN region and having different lengthsencompassing successively larger portions (411, 413, 415) of V region(402). Members of a nested set may be grouped together after sequencingby noting the identify (or substantial identity) of their respectiveNDN, J and/or C regions, thereby allowing reconstruction of a longerV(D)J segment than would be the case otherwise for a sequencing platformwith limited read length and/or sequence quality. In one embodiment, theplurality of primer sets may be a number in the range of from 2 to 5. Inanother embodiment the plurality is 2-3; and still another embodimentthe plurality is 3. The concentrations and positions of the primers in aplurality may vary widely. Concentrations of the V region primers may ormay not be the same. In one embodiment, the primer closest to the NDNregion has a higher concentration than the other primers of theplurality, e.g. to insure that amplicons containing the NDN region arerepresented in the resulting amplicon. One or more primers (e.g. 435 and437 in FIG. 4B) adjacent to the NDN region (444) may be used to generateone or more sequence reads (e.g. 434 and 436) that overlap the sequenceread (442) generated by J region primer (432), thereby improving thequality of base calls in overlap region (440). Sequence reads from theplurality of primers may or may not overlap the adjacent downstreamprimer binding site and/or adjacent downstream sequence read. In oneembodiment, sequence reads proximal to the NDN region (e.g. 436 and 438)may be used to identify the particular V region associated with theclonotype. Such a plurality of primers reduces the likelihood ofincomplete or failed amplification in case one of the primer bindingsites is hypermutated during immunoglobulin development. It alsoincreases the likelihood that diversity introduced by hypermutation ofthe V region will be capture in a clonotype sequence. A secondary PCRmay be performed to prepare the nested amplicons for sequencing, e.g. byamplifying with the P5 (401) and P7 (404, 406, 408) primers asillustrated to produce amplicons (420, 422, and 424), which may bedistributed as single molecules on a solid surface, where they arefurther amplified by bridge PCR, or like technique.

Base calling in NDN regions (particularly of IgH chains) can be improvedby using the codon structure of the flanking J and V regions, asillustrated in FIG. 4C. As used herein, “codon structure” means thecodons of the natural reading frame of segments of TCR or BCRtranscripts or genes outside of the NDN regions, e.g. the V region, Jregion, or the like.) There amplicon (450), which is an enlarged view ofthe amplicon of FIG. 4B, is shown along with the relative positions of Cread (442) and adjacent V read (434) above and the codon structures (452and 454) of V region (430) and J region (446), respectively, below. Inaccordance with this aspect of the invention, after the codon structures(452 and 454) are identified by conventional alignment to the V and Jreference sequences, bases in NDN region (456) are called (oridentified) one base at a time moving from J region (446) toward Vregion (430) and in the opposite direction from V region (430) toward Jregion (446) using sequence reads (434) and (442). Under normalbiological conditions, only the recombined TCR or IgH sequences thathave in frame codons from the V region through the NDN region and to theJ region are expressed as proteins. That is, of the variants generatedsomatically the only ones expressed are those whose J region and Vregion codon frames are in-frame with one another and remain in-framethrough the NDN region. (Here the correct frames of the V and J regionsare determined from reference sequences). If an out-of-frame sequence isidentified based one or more low quality base calls, the correspondingclonotype is flagged for re-evaluation or as a potential disease-relatedanomaly. If the sequence identified is in-frame and based on highquality base calls, then there is greater confidence that thecorresponding clonotype has been correctly called. Accordingly, in oneaspect, the invention includes a method of determining V(D)J-basedclonotypes from bidirectional sequence reads comprising the steps of:(a) generating at least one J region sequence read that begins in a Jregion and extends into an NDN region and at least one V region sequenceread that begins in the V regions and extends toward the NDN region suchthat the J region sequence read and the V region sequence read areoverlapping in an overlap region, and the J region and the V region eachhave a codon structure; (b) determining whether the codon structure ofthe J region extended into the NDN region is in frame with the codonstructure of the V region extended toward the NDN region. In a furtherembodiment, the step of generating includes generating at least one Vregion sequence read that begins in the V region and extends through theNDN region to the J region, such that the J region sequence read and theV region sequence read are overlapping in an overlap region.

Analyzing Sequence Reads. Coalescing sequence reads into clonotypes.Constructing clonotypes from sequence read data depends in part on thesequencing method used to generate such data, as the different methodshave different expected read lengths and data quality. In one approach,a Solexa sequencer is employed to generate sequence read data foranalysis. In one embodiment, a sample is obtained that provides at least0.5-1.0×10⁶ lymphocytes to produce at least 1 million templatemolecules, which after optional amplification may produce acorresponding one million or more clonal populations of templatemolecules (or clusters). For most high throughput sequencing approaches,including the Solexa approach, such over sampling at the cluster levelis desirable so that each template sequence is determined with a largedegree of redundancy to increase the accuracy of sequence determination.For Solexa-based implementations, preferably the sequence of eachindependent template is determined 10 times or more. For othersequencing approaches with different expected read lengths and dataquality, different levels of redundancy may be used for comparableaccuracy of sequence determination. Those of ordinary skill in the artrecognize that the above parameters, e.g. sample size, redundancy, andthe like, are design choices related to particular applications.

Reducing a set of reads for a given sample into its distinct clonotypesand recording the number of reads for each clonotype would be a trivialcomputational problem if sequencing technology was error free. However,in the presence of sequencing errors, each clonotype is surrounded by a‘cloud’ of reads with varying numbers of errors with respect to the trueclonotype sequence. The higher the number of such errors the smaller thedensity if the surrounding cloud, i.e. the cloud drops off in density aswe move away from the clonotype in sequence space. A variety ofalgorithms are available for converting sequence reads into clonotypes.In one aspect, coalescing of sequence reads depends on three factors:the number of sequences obtained for each of the two clonotypes ofinterest; the number of bases at which they differ; and the sequencingquality at the positions at which they are discordant. A likelihoodratio is assessed that is based on the expected error rates and binomialdistribution of errors. For example two clonotypes, one with 150 readsand the other with 2 reads with one difference between them in an areaof poor sequencing quality will likely be coalesced as they are likelyto be generated by sequencing error. On the other hand two clonotypes,one with 100 reads and the other with 50 reads with two differencesbetween them are not coalesced as they are considered to be unlikely tobe generated by sequencing error. In one embodiment of the invention,the algorithm described below may be used for determining clonotypesfrom sequence reads.

This cloud of reads surrounding each clonotype can be modeled using thebinomial distribution and a simple model for the probability of a singlebase error. This latter error model can be inferred from mapping V and Jsegments or from the clonotype finding algorithm itself, viaself-consistency and convergence. A model is constructed for theprobability of a given ‘cloud’ sequence Y with read count C2 and Eerrors with respect to sequence X) being part of a true clonotypesequence X with perfect read count C1 under the null model that X is theonly true clonotype in this region of sequence space. A decision is madewhether or not to coalesce sequence Y into the clonotype X according theparameters C1, C2, and E. For any given C1 and E a max value C2 ispre-calculated for deciding to coalesce the sequence Y. The max valuesfor C2 are chosen so that the probability of failing to coalesce Y underthe null hypothesis that Y is part of clonotype X is less than somevalue P after integrating over all possible sequences Y with error E inthe neighborhood of sequence X. The value P is controls the behavior ofthe algorithm and makes the coalescing more or less permissive.

If a sequence Y is not coalesced into clonotype X because its read countis above the threshold C2 for coalescing into clonotype X then itbecomes a candidate for seeding separate clonotypes. The algorithm alsomakes sure than any other sequences Y2, Y3, etc. which are ‘nearer’ tothis sequence Y that had been deemed independent of X) are notaggregated into X. This concept of ‘nearness’ includes both error countswith respect to Y and X and the absolute read count of X and Y, i.e. itis modeled in the same fashion as the above model for the cloud of errorsequences around clonotype X. In this way ‘cloud’ sequences can beproperly attributed to their correct clonotype if they happen to be‘near’ more than one clonotype.

The algorithm proceeds in a top down fashion by starting with thesequence X with the highest read count. This sequence seeds the firstclonotype. Neighboring sequences are either coalesced into thisclonotype if their counts are below the precalculated thresholds (seeabove), or left alone if they are above the threshold or ‘closer’ toanother sequence that was not coalesced. After searching all neighboringsequences within a maximum error count, the process of coalescing readsinto clonotype X is finished. Its reads and all reads that have beencoalesced into it are accounted for and removed from the list of readsavailable for making other clonotypes. The next sequence is then movedon to with the highest read count. Neighboring reads are coalesced intothis clonotype as above and this process is continued until there are nomore sequences with read counts above a given threshold, e.g. until allsequences with more than 1 count have been used as seeds for clonotypes.

In another embodiment of the above algorithm, a further test may beadded for determining whether to coalesce a candidate sequence Y into anexisting clonotype X, which takes into account quality score of therelevant sequence reads. The average quality score(s) are determined forsequence(s) Y (averaged across all reads with sequence Y) were sequencesY and X differ. If the average score is above a predetermined value thenit is more likely that the difference indicates a truly differentclonotype that should not be coalesced and if the average score is belowsuch predetermined value then it is more likely that sequence Y iscaused by sequencing errors and therefore should be coalesced into X.

Sequence Tree. The above algorithm of coalescing reads into clonotypesis dependent upon having an efficient way of finding all sequences withless than E errors from some input sequence X. This problem is solvedusing a sequence tree. The implementation of this tree has some unusualfeatures in that the nodes of the tree are not restricted to beingsingle letters of DNA. The nodes can have arbitrarily long sequences.This allows for a more efficient use of computer memory.

All of the reads of a given sample are placed into the sequence tree.Each leaf nodes holds pointers to its associated reads. It correspondsto a unique sequence given by traversing backwards in the tree from theleaf to the root node. The first sequence is placed into a simple treewith one root node and one leaf node that contains the full sequence ofthe read. Sequences are next added one by one. For each added sequenceeither a new branch is formed at the last point of common sequencebetween the read and the existing tree or add the read to an existingleaf node if the tree already contains the sequence.

Having placed all the reads into the tree it is easy to use the tree forthe following purposes: 1. Highest read count: Sorting leaf nodes byread count allows us to find the leaf node (i.e. sequence) with the mostreads. 2. Finding neighboring leafs: for any sequence all paths throughthe tree which have less than X errors with respect to this sequence aresearchable. A path is started at the root and branch this path intoseparate paths proceeding along the tree. The current error count ofeach path as proceeding along the tree is noted. When the error countexceeds the max allowed errors the given path is terminated. In this waylarge parts of the tree are pruned as early as possible. This is anefficient way of finding all paths (i.e. all leafs) within X errors fromany given sequence.

Somatic Hypermutations. In one embodiment, IgH-based clonotypes thathave undergone somatic hypermutation are determined as follows. Asomatic mutation is defined as a sequenced base that is different fromthe corresponding base of a reference sequence (of the relevant segment,usually V, J or C) and that is present in a statistically significantnumber of reads. In one embodiment, C reads may be used to find somaticmutations with respect to the mapped J segment and likewise V reads forthe V segment. Only pieces of the C and V reads are used that wereeither directly mapped to J or V segments or that were inside theclonotype extension up to the NDN boundary. In this way, the NDN regionis avoided and the same ‘sequence information’ is not used for mutationfinding that was previously used for clonotype determination (to avoiderroneously classifying as mutations nucleotides that are really justdifferent recombined NDN regions). For each segment type, the mappedsegment (major allele) is used as a scaffold and all reads areconsidered which have mapped to this allele during the read mappingphase. Each position of the reference sequences where at least one readhas mapped is analyzed for somatic mutations. In one embodiment, thecriteria for accepting a non-reference base as a valid mutation includethe following: 1) at least N reads with the given mutation base, 2) atleast a given fraction N/M reads (where M is the total number of mappedreads at this base position) and 3) a statistical cut based on thebinomial distribution, the average Q score of the N reads at themutation base as well as the number (M−N) of reads with a non-mutationbase. Preferably, the above parameters are selected so that the falsediscovery rate of mutations per clonotype is less than 1 in 1000, andmore preferably, less than 1 in 10000.

Phylogenic Clonotypes (Clans). In some diseases, such as cancers,including lymphoid proliferative disorders, a single lymphocyteprogenitor may give rise to many related lymphocyte progeny, eachpossessing and/or expressing a slightly different TCR or BCR, andtherefore a different clonotype, due to on-going somatic hypermutationor to disease-related somatic mutation(s), such as base substitutions,aberrant rearrangements, or the like. Cells producing such clonotypesare referred to herein as phylogenic clones, and a set of such relatedclones are referred to herein as a “clan.” Likewise, clonotypes ofphylogenic clones are referred to as phylogenic clonotypes and a set ofphylogenic clonotypes may be referred to as a clan of clonotypes. In oneaspect, methods of the invention comprise monitoring the frequency of aclan of clonotypes (i.e., the sum of frequencies of the constituentphylogenic clonotypes of the clan), rather than a frequency of anindividual clonotype. (The expression “one or more patient-specificclonotypes” encompasses the concept of clans). Phylogenic clonotypes maybe identified by one or more measures of relatedness to a parentclonotype. In one embodiment, phylogenic clonotypes may be grouped intothe same clan by percent homology, as described more fully below. Inanother embodiment, phylogenic clonotypes are identified by common usageof V regions, J regions, and/or NDN regions. For example, a clan may bedefined by clonotypes having common J and ND regions but different Vregions sometimes referred to as “VH replacement”); or it may be definedby clonotypes having the same V and J regions (identically mutated bybase substitutions from their respective reference sequences) but withdifferent NDN regions; or it may be defined by a clonotype that hasundergone one or more insertions and/or deletions of from 1-10 bases, orfrom 1-5 bases, or from 1-3 bases, to generate clan members. In anotherembodiment, clonotypes are assigned to the same clan if they satisfy thefollowing criteria: i) they are mapped to the same V and J referencesegments, with the mappings occurring at the same relative positions inthe clonotype sequence, and ii) their NDN regions are substantiallyidentical. “Substantial” in reference to clan membership means that somesmall differences in the NDN region are allowed because somaticmutations may have occurred in this region. Preferably, in oneembodiment, to avoid falsely calling a mutation in the NDN region,whether a base substitution is accepted as a cancer-related mutationdepends directly on the size of the NDN region of the clan. For example,a method may accept a clonotype as a clan member if it has a one-basedifference from clan NDN sequence(s) as a cancer-related mutation if thelength of the clan NDN sequence(s) is m nucleotides or greater, e.g. 9nucleotides or greater, otherwise it is not accepted, or if it has atwo-base difference from clan NDN sequence(s) as cancer-relatedmutations if the length of the clan NDN sequence(s) is n nucleotides orgreater, e.g. 20 nucleotides or greater, otherwise it is not accepted,In another embodiment, members of a clan are determined using thefollowing criteria: (a) V read maps to the same V region, (b) C readmaps to the same J region, (c) NDN region substantially identical (asdescribed above), and (d) position of NDN region between V-NDN boundaryand J-NDN boundary is the same (or equivalently, the number ofdownstream base additions to D and the number of upstream base additionsto D are the same). As used herein, the term “C read” may refer to aread generated from a sequencing primer that anneals either to a Cregion (in the case of using an RNA sample) or to a J region (in thecase of using a DNA sample). As explained else where, this is because aC region is joined with a J region in a post-transcriptional splicingprocess.

Phylogenic clonotypes of a single sample may be grouped into clans andclans from successive samples acquired at different times may becompared with one another. In particular, in one aspect of theinvention, clans containing clonotypes correlated with a disease, suchas a lymphoid neoplasm, are identified among clonotypes determined fromeach samples at each time point. The set (or clan) of correlatingclonotypes from each time point is compared with that of the immediatelyprevious sample to determine disease status by, for example, determiningin successive clans whether a frequency of a particular clonotypeincreases or decreases, whether a new correlating clonotype appears thatis known from population studies or databases to be correlating, or thelike. A determined status could be continued remission, incipientrelapse, evidence of further clonal evolution, or the like.

Isotype usage. In a further aspect, the invention provides clonotypeprofiles that include isotype usage information. Whenever IgH- orTCRf3-based clonotypes are determined from RNA, post-transcriptionalsplicing joins C regions to J regions, as illustrated in FIG. 3B. In oneaspect, sequencing primers used to generate C reads (e.g. 304) areanneal to a predetermined primer binding site (302) in C region (307) atthe junction with J region (309). If primer binding site (302) isselected so that C read (304) includes a portion (305) of C region(307), then the identity of C region (307) may be determined which, inturn, permits the isotype of the synthesized BCR to be determined. Inone embodiment, primer binding site (302) is selected so that C read(304) includes at least six nucleotides of C region (307); in anotherembodiment, primer binding site (302) is selected so that C read (304)includes at least 8 nucleotides of C region (307). Each clonotypedetermined in accordance with this embodiment includes sequenceinformation from portion (305) of its corresponding C region and fromsuch sequence information its corresponding isotype is determined. Inone aspect of the invention, correlating clonotypes may have a firstisotype at the time they are initially determined, but may switch toanother type of isotype during the time they are being monitored. Thisembodiment is capable of detecting such switches by noting previouslyunrecorded clonotypes that have identical sequences to the correlatingclonotypes, except for the sequence of portion (305) which correspondsto a different isotype.

It is expected that PCR error is concentrated in some bases that weremutated in the early cycles of PCR. Sequencing error is expected to bedistributed in many bases even though it is totally random as the erroris likely to have some systematic biases. It is assumed that some baseswill have sequencing error at a higher rate, say 5% (5 fold theaverage). Given these assumptions, sequencing error becomes the dominanttype of error. Distinguish PCR errors from the occurrence of highlyrelated clonotypes will play a role in analysis. Given the biologicalsignificance to determining that there are two or more highly relatedclonotypes, a conservative approach to making such calls is taken. Thedetection of enough of the minor clonotypes so as to be sure with highconfidence (say 99.9%) that there are more than one clonotype isconsidered. For example of clonotypes that are present at 100copies/1,000,000, the minor variant is detected 14 or more times for itto be designated as an independent clonotype. Similarly, for clonotypespresent at 1,000 copies/1,000,000 the minor variant can be detected 74or more times to be designated as an independent clonotype. Thisalgorithm can be enhanced by using the base quality score that isobtained with each sequenced base. If the relationship between qualityscore and error rate is validated above, then instead of employing theconservative 5% error rate for all bases, the quality score can be usedto decide the number of reads that need to be present to call anindependent clonotype. The median quality score of the specific base inall the reads can be used, or more rigorously, the likelihood of beingan error can be computed given the quality score of the specific base ineach read, and then the probabilities can be combined (assumingindependence) to estimate the likely number of sequencing error for thatbase. As a result, there are different thresholds of rejecting thesequencing error hypothesis for different bases with different qualityscores. For example for a clonotype present at 1,000 copies per1,000,000 the minor variant is designated independent when it isdetected 22 and 74 times if the probability of error were 0.01 and 0.05,respectively.

III. Correlating Clonotypes and Medical Algorithms

The invention provides methods for identifying clonotypes whosepresence, absence and/or level is correlated to a disease state and forusing such information to make diagnostic or prognostic decisions. Inone aspect, information from clonotype profiles, which may be coupledwith other medical information, such as expression levels of non-TCR ornon-BCR genes, physiological condition, or the like, is presented topatients or healthcare providers in the context of an algorithm; thatis, a set of one or more steps in which results of tests and/orexaminations are assessed and (i) either a course of action isdetermined or a decision as to health or disease status is made or (ii)a series of decisions are made in accordance with a flow chart, or likedecision-making structure, that leads to a course of action, or adecision as to health or disease status. Algorithms of the invention mayvary widely in format. For example, an algorithm may simply suggest thata patient should be treated with a drug, if a certain clonotype, orsubset of clonotypes, exceeds a predetermined ratio in a clonotypeprofile, or increases in proportion at more than a predetermined ratebetween monitoring measurements. Even more simply, an algorithm maymerely indicate that a positive correlation exist between a diseasestatus and a level of one or more clonotypes and/or a function of TCRsor BCRs encoded by one or more clonotypes. More complex algorithms mayinclude patient physiological information in addition to informationfrom one or more clonotype profiles. For example, in complex disorders,such as some autoimmune disorders, clonotype profile information may becombined in an algorithm with other patient data such as prior course oftreatment, presence, absence or intensity of symptoms, e.g. rash, jointinflammation, expression of particular genes, or the like. In one aspectof the invention, an algorithm for use with monitoring lymphoiddisorders provides a predetermined fractional value above which theproportion of a clonotype (and/or evolutionarily related clonotypes) ina clonotype profile of a sample (such as a blood sample) indicates arelapse of disease or a resistance to a treatment. Such algorithms mayconsist of or include conventional measures of TCR or BCR clonality. Inanother aspect, an algorithm for use with monitoring autoimmunedisorders provides one or more predetermined fractional values abovewhich a proportion of clonotypes in a clonotype profile encoding TCRs orBCRs specific for one or more predetermined antigens, respectively,indicates the onset of an autoimmune flare-up.

A. Correlating Versus Non-Correlating Clonotypes

The methods of the present invention provide means for distinguishing a)correlating clonotypes (which can be those clonotypes whose levelcorrelate with disease) from b) non-correlating clonotypes (which can bethose clonotypes whose levels do not correlate with disease). In oneembodiment, a correlating clonotype can display either positive ornegative correlation with disease. In another embodiment, a clonotypepresent at a peak state of a disease but not present at a non-peak stateof a disease can be a correlating clonotype (positive correlation withdisease). In another embodiment, a clonotype that is more abundant (i.e.is present at a higher level of molecules) in a peak state (or stage) ofa disease than at a non-peak state of the disease can be a correlatingclonotype (positive correlation with the disease). In anotherembodiment, a clonotype absent at a peak state of a disease but presentduring a non-peak state of the disease can be a correlating clonotype(negative correlation with disease). In another embodiment, a clonotypethat is less abundant at a peak state of a disease than at a non-peakstate of a disease can be a correlating clonotype (negative correlationwith disease). In another embodiment, a correlating clonotype for anindividual is determined by an algorithm.

B. Discovering Correlating and Non-Correlating Clonotypes Using aCalibration Test without a Population Study.

In one embodiment of the invention, correlating clonotypes areidentified by looking at the clonotypes present in some sample that hasrelevance to a disease state. This sample could be blood from a sampleat a peak state of disease (e.g. a blood sample from an MS or lupuspatient during an acute flare), or it could be from a disease-affected,or disease-related, tissue, that is enriched for T and B cells involvedin the disease for that individual, such as an inflammation or tumor.Examples of these tissues could be kidney biopsies of lupus patientswith kidney inflammations, cerebral spinal fluid (CSF) in MS patientsduring a flare, synovial fluid for rheumatoid arthritis patients, ortumor samples from cancer patients. In all of these examples, it islikely that the tissues will contain relevant T and B cells that arerelated to the disease (though not necessarily the causative agents). Itis notable that if this method is used to identify the clonotypes thatare relevant to disease, they will only be relevant to the individual inwhose sample they were detected. As a result, a specific calibrationtest is needed in order to use this method to identify correlatingclonotypes in any given individual with a disease. That is, in oneaspect, correlating clonotypes are discovered or determined bygenerating a clonotype profile from a sample taken from a tissuedirectly affected by, or relevant to, a disease (sometimes referred toherein as a “disease-related tissue”). In a further aspect, suchdetermination further includes generating a clonotype profile from asample taken from a tissue not affected by, or relevant to, a disease(sometimes referred to herein as a “non-disease-related tissue”), thencomparing the former and latter clonotype profiles to identifycorrelating clonotypes as those that are at a high level, low level orthat are functionally distinct, e.g. encode TCRs or BCRs specific for aparticular antigen. In one aspect, such determination is made byidentifying clonotypes present in a clonotype profile from an affected,or disease-related, tissue at a higher frequency than the sameclonotypes in a clonotype profile of non-affected, ornon-disease-related, tissue.

In one embodiment, a method for determining one or more correlatingclonotypes in a subject is provided. The method can include steps for a)generating one or more clonotype profiles by nucleic acid sequencingindividual, spatially isolated molecules from at least one sample fromthe subject, wherein the at least one sample is related to a first stateof the disease, and b) determining one or more correlating clonotypes inthe subject based on the one or more clonotype profiles.

In one embodiment, at least one sample is from a tissue affected by thedisease. In another embodiment, said determination of one or morecorrelating clonotypes comprises comparing clonotype profiles from atleast two samples. In another embodiment, the first state of the diseaseis a peak state of the disease. In another embodiment, one or morecorrelating clonotypes are present in the peak state of the disease. Inanother embodiment, the one or more correlating clonotypes are absent inthe peak state of the disease. In another embodiment, one or morecorrelating clonotypes are high in the peak state of the disease. Inanother embodiment, one or more correlating clonotypes are low in thepeak state of the disease. In another embodiment, the sample comprisesT-cells and/or B-cells. In another embodiment, the T-cells and/orB-cells comprise a subset of T-cells and/or B-cells. In anotherembodiment, the subset of T-cells and/or B-cells are enriched byinteraction with a marker. In another embodiment, the marker is a cellsurface marker on the subset of T-cells and/or B-cells. In anotherembodiment, the subset of T-cells and/or B-cells interacts with anantigen specifically present in the disease. For example, in the case oflymphoproliferative disorders, such as lymphomas, a calibrating samplemay be obtained from lymphoid tissues, from lesions caused by thedisorder, e.g. metastatic lesions, or from tissues indirectly affectedby the disorder by enrichment as suggested above. For lymphoid neoplasmsthere is widely available guidance and commercially available kits forimmunophenotyping and enriching disease-related lymphocytes, e.g.“U.S.-Canadian consensus recommendations on the immunophenotypicanalysis of haematologic neoplasia by flow cytometry,” Cytometry, 30:214-263 (1997); MultiMix™ Antibody Panels for Immunophenotyping Leukemiaand Lymphoma by Flow Cytometry (Dako, Denmark); and the like. Lymphoidtissues include lymph nodes, spleen, tonsils, adenoids, thymus, and thelike.

In one embodiment, the disease is an autoimmune disease. In anotherembodiment, the autoimmune disease is systemic lupus erythematosus,multiple sclerosis, rheumatoid arthritis, or Ankylosing Spondylitis.

In some embodiments, the correlating clonotypes are identified bylooking at the clonotypes present in some sample that has relevance to astate other than a disease state. These states could include exposure tonon-disease causing antigens, such as sub-symptomatic allergic reactionsto local pollens. Such an embodiment could be used to identify whetheran individual had recently returned to a geography which contained theantigen. The states could include exposure to an antigen related to anindustrial process or the manufacture or production of bioterrorismagents.

C. Discovering Correlating and Non-Correlating Clonotypes Using aPopulation Study.

In one embodiment, a method is provided for identifying correlatingclonotypes using a population study. The utility of the population studyis that it allows the specific information about correlating clonotypesthat have been ascertained in individuals with known disease stateoutcomes to be generalized to allow such correlating clonotypes to beidentified in all future subjects without the need for a calibrationtest. Knowledge of a specific set of correlating clonotypes can be usedto extract rules about the likely attributes (parameters) of clonotypesthat will correlate in future subjects. Such embodiment is implementedwith the following steps: (a) generating clonotype profiles for each ofa set of samples from tissues affected by, or relevant to, a disease;(b) determining clonotypes that are at a high level or low levelrelative to the same clonotypes in samples from non-affected tissues orthat are functionally distinct from clonotypes in samples fromnon-affected tissues. As used herein, in one aspect, “functionallydistinct” in reference to clonotypes means that TCRs or BCRs encoded byone are specific for a different antigen, protein or complex than theother. Optionally, the above embodiment may further include a step ofdeveloping an algorithm for predicting correlating clonotypes in anysample from the sequence information of the clonotypes determined inabove steps (a) and/or (b) or from the functional data, i.e. adetermination that the newly measured clonotypes encode TCRs or BCRsspecific for an antigen, protein or complex specific for the diseaseunder observation.

In connection with the above, one or more patient-specific clonotypesmay be identified by matching clonotypes determined in one or moreinitial measurements “determined clonotypes”) with clonotypes known tobe correlated with said disease, which may be available through apopulation study, database, or the like. In one aspect, matching suchclonotypes comprises finding identity between an amino acid sequenceencoded by the determined clonotype and that of an amino acid sequenceencoded by a clonotype known to be correlated to the disease, or asubstantially identical variant the latter clonotype. As used herein,“substantially identical variant”, in one aspect, means the sequencesbeing compared or matched are at least 80 percent identical, or at least90 percent identical, whether nucleic acid sequence or amino acidsequence. In another aspect, substantially identical variant meansdiffering by 5 or less base or amino acid additions, deletions and/orsubstitutions. In another aspect, matching such clonotypes comprisesfinding identity between the determined clonotype and a nucleic acidsequence of a clonotype known to be correlated to the disease, or asubstantially identical variant of the latter clonotype. In stillanother aspect, matching such clonotypes comprises finding identitybetween the determined clonotype and a nucleic acid sequence of aclonotype known to be correlated to the disease, or a substantiallyidentical variant of the latter clonotype.

In one embodiment, the provided invention encompasses methods thatinclude identifying correlating and non-correlating clonotypes bysequencing the immune cell repertoire in a study of samples frompatients with disease(s) and optionally healthy controls at differenttimes and, in the case of the patients with a disease, at different (andknown) states of the disease course characterized by clinical data. Thedisease can be, for example, an autoimmune disease. The clonotypes whoselevel is correlated with measures of disease in these different statescan be used to develop an algorithm that predicts the identity of alarger set of sequences that will correlate with disease as distinctfrom those that will not correlate with disease in all individuals.Unlike the case of the calibration test, correlating sequences need nothave been present in the discovery study but can be predicted based onthese sequences. For example, a correlating sequence can be TCR gene DNAsequence that encodes the same amino acid sequence as the DNA sequenceof a clonotype identified in the discovery study. Furthermore, thealgorithm that can predict one or more correlating clonotypes can beused to identify clonotypes in a sample from any individual and is in noway unique to a given individual, thus allowing the correlatingclonotypes to be predicted in a novel sample without prior knowledge ofthe clonotypes present in that individual.

In one aspect, a method for developing an algorithm that predicts one ormore correlating clonotypes in any sample from a subject with a diseaseis provided comprising: a) generating a plurality of clonotype profilesfrom a set of samples, wherein the samples are relevant to the disease,b) identifying one or more correlating clonotypes from the set ofsamples, c) using sequence parameters and/or functional data from one ormore correlating clonotypes identified in b) to develop an algorithmthat can predict correlating clonotypes in any sample from a subjectwith the disease.

In one embodiment, the set of samples are taken from one or more tissuesaffected by the disease.

In another embodiment, the identifying one or more correlatingclonotypes comprises comparing clonotype profiles from at least twosamples. In another embodiment, the functional data include bindingability of markers in T-cell and/or B-cells or interaction with antigenby a T-cell or B cell. In another embodiment, said sequence parameterscomprise nucleic acid sequence and predicted amino acid sequence. Inanother embodiment, the samples are from one or more individuals at apeak stage of the disease. In another embodiment, said one or morecorrelating clonotypes are present in the peak state of the disease. Inanother embodiment, said one or more correlating clonotypes are at ahigh level in the peak state of the disease. In another embodiment, oneor more correlating clonotypes are at a low level in the peak state ofthe disease. In another embodiment, one or more correlating clonotypesare absent at the peak state of the disease.

In one embodiment, the disease is an autoimmune disease. In anotherembodiment, the autoimmune disease is systemic lupus erythematosus,multiple sclerosis, rheumatoid arthritis, or Ankylosing Spondylitis.

In another aspect, a method for discovering one or more correlatingclonotypes for an individual is provided, comprising a) inputting aclonotype profile from a sample from the individual into an algorithm,and b) using the algorithm to determine one or more correlatingclonotypes for the individual. The algorithm can be an algorithmdeveloped by: a) generating a plurality of clonotype profiles from a setof samples, wherein the samples are relevant to the disease, b)identifying one or more correlating clonotypes from the set of samples,and c) using sequence parameters and/or functional data from one or morecorrelating clonotypes identified in b) to develop the algorithm thatcan predict correlating clonotypes in any sample from a subject with thedisease.

In some embodiments, the correlating clonotypes are identifiedclonotypes present in populations that have been exposed to an antigenwhich has relevance to a state other than a disease state. This statecould include exposure to non-disease causing antigens, such assub-symptomatic allergic reactions to local pollens. Such an embodimentcould be used to identify whether an individual had recently traveled toa geography which contained the antigen. The states could includeexposure to an antigen related to an industrial process or themanufacture or production of bioterrorism agents.

D. Discovering Correlating and Non-Correlating Clonotypes Using aCalibration Test Combined with a Population Study.

In one embodiment of the invention the correlating clonotypes areidentified by using a calibration test combined with a population study.In this embodiment the population study does not result in an algorithmthat allows clonotypes to be predicted in any sample but rather itallows an algorithm to be developed to predict correlating clonotypes inany sample from a subject for whom a particular calibration clonotypeprofile has been generated. An example of this could be the developmentof an algorithm that would predict the correlating clonotypes in a lupuspatient based on the clonotype profile measured from a blood sample atany stage of disease after having first having had a blood test takenduring a clinical flare state that was used to calibrate the algorithm.Thus, in this embodiment, correlating clonotypes may be identified insteps: (a) generating clonotype profiles from a set of samples fromtissues relevant to or affected by a disease to identify a set ofclonotypes associated with the disease either by level and/or byfunction and to identify a relationship between such level and/orfunction and disease status; (b) measuring a clonotype profile of asample from a tissue of a first state of the disease; (c) determining acorrelating clonotype from the relationship of step (a). In anotherembodiment, correlating clonotypes may be identified in steps: (a)generating clonotype profiles from a set of samples from tissuesrelevant to or affected by a disease to identify a set of clonotypesassociated with the disease either by level and/or by function and toidentify a relationship between such level and/or function and diseasestatus; (b) measuring a calibration clonotype profile in a new subjectat a relevant disease stage at a peak stage or from disease affectedtissue or at a functionally characterized state; (c) determining acorrelating clonotype from the relationship of step (a).

In this embodiment the provided invention encompasses methods foridentifying correlating and non-correlating clonotypes by sequencing theimmune cell repertoire in a study of samples from patients of disease(s)and optionally healthy controls at different times and, in the case ofthe patients with a disease, at different (and known) states of thedisease course characterized by clinical data. The clonotypes that arefound at different frequency (or level) in the first state than in thesecond state are then used to develop an algorithm that predicts whichof the sequences found in the repertoires of each individual at thefirst disease state will correlate with disease at the later state ineach individual as distinct from those that will not correlate withdisease in that individual. Unlike the case of the calibration testalone, correlating sequences may be a subset of all the sequences foundto be different between disease states. It is also possible thatcorrelating clonotypes are not found in the calibration sample but arepredicted based on the algorithm to be correlating if they appear in afuture sample. As an example, a clonotype that codes for the same aminoacid sequence as a clonotype found in a calibration sample may bepredicted to be a correlating clonotype based on the algorithm thatresults from the population study. Unlike the previous embodiments, thealgorithm is developed to predict the correlating clonotypes based on acalibration clonotype profile which is a clonotype profile generated inthe individual for whom the correlating clonotypes are to be predictedwhich at a specific state of disease. In this embodiment the algorithmcannot be used to generate correlating clonotypes in a particularindividual until a specific calibration clonotype profile has beenmeasured. After this calibration profile has been measured in aparticular subject, all subsequent correlating clonotypes can bepredicted based on the measurement of the clonotype profiles in thatindividual.

In another aspect, a method for discovering one or more correlatingclonotypes for an individual is provided, comprising a) inputting aclonotype profile from a sample from the individual into an algorithm,and b) using the algorithm to determine one or more correlatingclonotypes for the individual. The algorithm can be an algorithmdeveloped by: a) generating a plurality of clonotype profiles from a setof samples, wherein the samples are relevant to the disease, b)identifying one or more correlating clonotypes from the set of samples,and c) using sequence parameters and/or functional data from one or morecorrelating clonotypes identified in b) to develop an algorithm that canpredict correlating clonotypes in any sample from a subject with thedisease. In one embodiment, the sample is at taken at a peak state ofdisease. In another embodiment, the sample is taken from diseaseaffected tissue.

In some embodiments, correlating and non-correlating clonotypes using acalibration test combined with a population study is performed forclonotypes present in populations that have been exposed to an antigenwhich has relevance to a state other than a disease state. This statecould include exposure to non-disease causing antigens, such assub-symptomatic allergic reactions to local pollens. Such an embodimentcould be used to identify whether an individual had recently traveled toa geography which contained the antigen. The states could includeexposure to an antigen related to an industrial process or themanufacture or production of bioterrorism agents.

E1. Sequence Related Parameters that can be Used to Predict CorrelatingClonotypes

In order to conduct a population study a training set can be used tounderstand the characteristics of correlating clonotypes by testingvarious parameters that can distinguish those correlating clonotypesfrom those that do not. These parameters include the sequence or thespecific V, D, and J segments used. In one embodiment it is shown thatspecific V segments are more likely to correlate with some diseases asis the case if the clonotypes for a specific disease are likely torecognize related epitopes and hence may have sequence similarity. Otherparameters included in further embodiments include the extent of somatichypermutation identified and the level of a clonotype at the peak of anepisode and its level when the disease is relatively inactive. Otherparameters that may predict correlating clonotypes include withoutlimitation: 1) sequence motifs including V or J region, a combinationVJ, short sequences in DJ region; 2) Sequence length of the clonotype;3) Level of the clonotype including absolute level (number of clones permillion molecules) or rank level; 4) Amino acid and nucleic acidsequence similarity to other clonotypes: the frequency of other highlyrelated clonotypes, including those with silent changes (nucleotidedifferences that code for same amino acids) or those with conservativeamino acid changes; 5) For the BCRs the level of somatic mutations inthe clonotype and/or the number of distinct clonotypes that differ bysomatic mutations from some germ line clonotypes; 6) clonotypes whoseassociated proteins have similar 3 dimensional structures.

E2. Databases of Clonotypes Encoding Antibodies Specific for an Antigen

This Correlating clonotypes may encode immunoglobulins or TCRs that arespecific for one or more epitopes of one or more antigens. Thus, in oneaspect of the invention, correlating clonotypes may be determined bycomparing measured clonotypes with entries of a database comprisingsubstantially all possible clonotypes to one or more selected antigensi.e. an “antigen-specific clonotype database”). Such databases may beconstructed by sequencing selected regions of antibody-encodingsequences of lymphocytes that produce antibodies with specificity forthe antigens or epitopes of interest, or such databases may be populatedby carrying out binding experiments with phage expressing and displayingantibodies or fragments thereof on their surfaces. The latter process isreadily carried out as described in Niro et al, Nucleic Acids Research,38(9): e110 (2010). Briefly, in one aspect, the method comprises thefollowing steps: (a) an antigen of interest, e.g. HCV core protein, isbound to a solid support, (b) a phage-encoded antibody library isexposed to the antigen under antibody-binding conditions so that afraction of phage-encoded antibodies binds to the bound antigen andanother fraction remains free, and (c) collecting and sequencing thephage-encoded antibodies that bind to create entries of a database ofcorrelating clonotypes. The bound phage-encoded antibodies areconveniently sequenced using a high-throughput DNA sequencing techniqueas described above. In one embodiment, clonotypes of the method encodesingle chain variable fragments (scFv) binding compounds.Antibody-binding conditions of different stringencies may be used. Thenucleic acid sequences determined from the bound phage may be tabulatedand entered into the appropriate antigen-specific clonotype database.

F. Functional Data to Refine the Determination of Correlating Clonotypes

Further embodiments will make use of functional data to aid inidentifying correlating clonotypes. For example, T-cells and/or B-cellscontaining certain markers that are enriched in cells containingcorrelating clonotypes can be captured through standard methods likeFACS or MACS. In another embodiment the marker is a cell-surface marker.In another embodiment T-cells and/or B-cells reactivity to an antigenrelevant to the pathology or to affected tissue would be good evidenceof the pathological relevance of a clonotype.

In another embodiment the sequence of the candidate clonotypes can besynthesized and put in the context of the full TCR or BCR and assessedfor the relevant reactivity. Alternatively, the amplified fragments ofthe different sequences can be used as an input to phage, ribosome, orRNA display techniques. These techniques can select for the sequenceswith the relevant reactivity. The comparison of the sequencing resultsfor those before and after the selection can identify those clones thathave the reactivity and hence are likely to be pathological. In anotherembodiment, the specific display techniques (for example phage,ribosome, or RNA display) can be used in an array format. The individualmolecules (or amplifications of these individual molecules) carryingindividual sequences from the TCR or BCR (for example CDR3 sequences)can be arrayed either as phages, ribosomes, or RNA. Specific antigenscan then be studied to identify the sequence(s) that code for peptidesthat bind them. Peptides binding antigens relevant to the disease arelikely to be pathological.

G. Generating an Immune Load Algorithm

An algorithm can be used to compute an Immune Load, a value or scoregiven by a function of the levels of correlating and non-correlatingclonotypes. The Immune Load can be used to make a clinical decision.Using data from an experiment, (e.g., an experiment comprising samplesfrom subjects in a first state of a disease and samples from subjects ina second state of the disease), an algorithm can be developed thatcombines the information about the levels of the correlating andnon-correlating clonotypes into a single score (Immune Load). Theparameters of this algorithm can then be adjusted to maximize thecorrelation between Immune Load and the clinical data. For example, theclinical data can be a clinical measure of disease severity (e.g., theextent of lesions on an MRI for a multiple sclerosis patient). Thus, inone embodiment, an Immune Load may be calculated by the steps: (a)developing an algorithm that uses a set of factors to combine the levelsof the correlating clonotypes into a single disease activity score; (b)comparing the score generated in step (a) to the clinical data aboutdisease state; and (c) optimizing the factors in order to maximize thecorrelation between the clinical data and the disease activity score.

The correlating clonotypes used in generating an Immune Load algorithmcan be generated using a calibration test, a population study, or acalibration test and a population study as described above.

Some of the factors that can be considered in combining the correlatingclonotypes are the number of correlating clonotypes, their level, theirrate of change (velocity), and the rate of change in the velocity(acceleration). Other factors to be assessed include the level of theclonotypes at the episode peak and at the inactive disease state

In one embodiment, the Immune Load generated relates to an autoimmunedisease. Such a Load can be referred to as an AutoImm Load.

In one aspect, a method for generating an algorithm that calculates adisease activity score is provided, comprising: a) developing analgorithm that uses a set of factors to combine levels of correlatingclonotypes into a disease activity score, b) comparing the diseaseactivity score to clinical data regarding the disease state, and c)optimizing the factors in order to maximize the correlation betweenclinical data and the disease activity score.

H. Monitoring Disease Using the Load Algorithm

1. Monitoring Disease without a Calibration Test

In one embodiment of the invention the clonotypes and the Immune Loadalgorithm are determined using a population study. Immune Load can beused directly without having to first calibrate the individual patient.This test can be done when the patient is in any disease state. Thistest can be used to generate specific correlating and non-correlatingclonotypes based on the algorithm developed above. Immune Load can thenbe calculated using the second algorithm generated in a populationstudy. This score can then be used clinically. In one embodiment, amonitoring test may be carried out without using a calibration test bythe following steps: (a) measuring the clonotypes of a patient at thetime at which the patient is to be monitored; and (b) using thecorrelating clonotypes predicted by the discovery algorithm test and thedata from the monitoring test to generate a score reflective of thedisease state of the patient using the monitoring algorithm.

In another aspect, a method for monitoring the disease state of anindividual is provided comprising: a) determining a clonotype profilefrom a sample from a subject, b) inputting the clonotype profileinformation from a) into an algorithm, and c) using the algorithm togenerate a score predictive of the disease state of the individual. Thealgorithm can be an algorithm generated by a) developing an algorithmthat uses a set of factors to combine levels of correlating clonotypesinto a disease activity score, b) comparing the disease activity scoreto clinical data regarding the disease state, and c) optimizing thefactors in order to maximize the correlation between clinical data andthe disease activity score.

2. Monitoring Disease Using a Calibration Test

In one embodiment of the provided invention the correlating clonotypesand the Immune Load algorithm are determined using a calibration test orcalibration test and a population study. Immune Load can be used in theclinic by first conducting a calibration test. This test can be donewhen the patient is in a state which is similar to the first state usedin the study that generated the correlating and non-correlatingclonotypes that are used in the Immune Load algorithm. For example, thisstate can be a flare state of an autoimmune disease if this is how theImmune Load algorithm was derived. This calibration test can then beused to generate the specific correlating and non-correlating clonotypesto be used in the subsequent disease monitoring tests. At a later pointin the treatment of this patient, another test is done on the patientand Immune Load can be calculated using the algorithm generated in thediscovery study, and the list of clonotype levels generated in thispatient's specific calibration test. This Immune Load score can then beused clinically. In one embodiment, a monitoring test using acalibration test comprises the following steps: (a) testing a patient indisease state 1 in order to determine a clonotype profile; (b) measuringclonotypes of a patient at a later time (the time at which the patientis to be monitored); (c) using the monitoring algorithm to generate adisease score reflective of a disease state from the clonotype profilefrom disease state 1 from the calibration test and information from alater time test.

In another aspect, a method for monitoring the disease state of anindividual is provided comprising: a) determining a clonotype profilefrom a sample from a subject, b) inputting the clonotype profileinformation from a) into an algorithm, and c) using the algorithm togenerate a score predictive of the disease state of the individual. Thealgorithm can be an algorithm generated a) developing an algorithm thatuses a set of factors to combine levels of correlating clonotypes into adisease activity score, b) comparing the disease activity score toclinical data regarding the disease state, and c) optimizing the factorsin order to maximize the correlation between clinical data and thedisease activity score. In another embodiment, the method can furthercomprise determining one or more correlating clonotypes in theindividual by any of the methods of the provided invention, andinputting information the one or more correlating clonotypes into thealgorithm.

In one embodiment, the disease is an autoimmune disease. In anotherembodiment, the autoimmune disease is systemic lupus erythematosus,multiple sclerosis, rheumatoid arthritis, or Ankylosing Spondylitis.

3. Other Factors Related to the Use of Immune Load

The same Immune Load may mean different things for different patients.For one, the full clinical picture of a patient needs to be considered.From a testing perspective, one may consider the velocity (rate ofchange of Immune Load over time) and acceleration (rate of change ofvelocity over time) in addition to the level of Immune Load in makingclinical decisions. For example if the AutoImm Load score is increasing(high velocity) it may be predictive of an incipient flare in anautoimmune disease.

Additional tests that can be integrated in the Load score, for example,an AutoImm Load score, include, for example, erythrocyte sedimentationrate (ESR), C-reactive protein (CRP) levels, Anti-ds DNA, otherautoantibody titers, complement levels, urine protein levels, Urineprotein/creatinine ratio, creatinine levels, blood urea nitrogen (BUN)levels, platelet levels, WBC counts, hematorcrit (Hct), Hb, urinalysisresults. Other tests that are related to SLE that can be integratedinclude, for example, CD27 level, CD27++ cell level, INF-responsivegenes (Baechler, E C et al. (2003) Proc. Natl. Acad. Sci. 100:2610-2615), and chemokine score (Bauer J W et al. (2009) ArthritisRheum. 60:3098-3107). Other tests not related to lupus include, forexample, thyroid-stimulating hormone (TSH) test, triiodothyronine (T3)test, thyroxine (T4) test, liver function tests (LFTs), otherautoantibodies, calprotectin test, lactoferrin test, and synovial fluidanalysis. The additional tests can include imaging test, including, forexample, MRI, CT-scan, X-ray, and ultrasound.

I. The Use of Sequencing Technologies in Combination with Partial CellEnrichments as Part of a Calibration Step to Find CorrelatingClonotypes.

Several technologies exist which can be used to separate cells in bloodor tissue based on cell markers. These include solid phase separationssuch as beads or columns on which specific affinity reagents areimmobilized such as antibodies. Liquid phase separation can be achievedusing such techniques as flow cytometry in which labeled reagents thatspecifically bind selected cell markers are used to direct a gated flowdevice in which specific fluorescent markers can be used to separatecells so labeled. Leukopheresis is another liquid phase separationtechnique that may be used to enrich leukocyte populations from theblood, e.g. Shelat, Am. J. Med. 123: 777-784 (2010); U.S. Pat. No.5,846,928; or the like, after which further enrichment may be carriedout by way of cell surface markers.

It will sometimes be advantageous to profile a subset of T and/or Bcells when calibrating an algorithm to predict correlating clonotypesand/or to measure the levels of these clonotypes in order to measure adisease load. This can be done using the methods described above for avariety of surface or internal markers. One challenge of using suchmethods lies in the fact that the selectivity for a given marker isnever perfect for a given marker. As a result enrichment is unlikely tolead to a pure population of the selected cells. Another method toachieve enrichment of cell subsets without the separation of cells is toselectively grow the subset of cells of interest. For example, T cellscan be activated with antigen in vitro, and the activated cells can beallowed to divide and increase in number leading to their enrichment.

In one embodiment of this invention, clonotype profiles of T and/or Bcell populations are generated by sequencing individual, spatiallyisolated molecules, both before and after enrichment for at least onecell marker. The two profiles are then compared to determine whichclonotypes have significantly altered frequencies between the enrichedand un-enriched cell populations in order to identify clonotypes thatare associated with cells carrying the markers selected for in theenrichment. The advantage of using the sequencing method inaccomplishing this identification is that clonotypes can be identifiedeven if the enrichment as relatively poor as clonotype frequencies canbe very well measured with sufficient sequencing depth. This in turnwill allow a variety of enrichment methodologies to be used routinelyand inexpensively as expensive multiple enrichments designed to achievepurity will not be necessary.

In one embodiment, such sequencing of T and B cell receptors before andafter cell enrichment could be used as part of a calibration algorithmin order to determine which clonotypes are correlating. In thisembodiment, cells are sequenced before and after enrichment for markersthat are relevant to disease at a first disease state. Examples of thisfirst disease state could be: blood samples at a peak state of thedisease, affected tissue samples, affected tissue samples at a peakstate of disease, etc. Clonotypes from the cell fraction that does anddoes not contain the cell marker are thus obtained and can be used asinputs into an algorithm that is then used to determine correlatingclonotypes in that individual.

In another embodiment, the enrichment is not only done on a samplecollected during a first disease state but in subsequent samples fromthe same individual. In this way, the correlating clonotypes aredetermined within sub fractions of the cells at any one time.

In another embodiment, cell markers are used in conjugation withsequencing to assess not only the frequency of specific relevantclonotypes but also their functional status. In this embodiment the cellmarkers provide information beyond the identity of the correlatingclonotypes as they refine the prediction of disease status obtained bythe frequency of the correlating clonotypes. By sequencing before andafter enrichment with specific markers, the frequency of a specificclonotype is determined. In addition the frequency and fraction of thecells containing this specific clonotype along with anther cell markeris determined Consider, two patients with the same frequency ofclonotypes relevant to some clinical state but different frequency ofparticular cell markers (e.g., activation markers) in the cellscontaining these clonotypes. These patients may have different diseaseactivity in spite of having the same frequency of the relevantclonotypes.

Cell markers can be markers of cell activation. In general markers canbe determined by measuring the expression of genes using a population ofT and/or B cells that are known to correlate with disease. These markerscould be cell surface markers or cells expressed within the cell.

In one embodiment of this invention, the cells that are shown to haveaffinity for an antigen known to be relevant to a particular disease areenriched. There are several methods for doing this.

In another embodiment, cells of interest are B cells that interact withspecific antigen. In this case, B cells will exist that have B-cellreceptor sequences that bind this specific antigen. These B cellreceptors can thus be used as cell surface markers that can be enrichedusing antigen specific reagents. In one embodiment, beads or columns onwhich the antigen is immobilized can be used to enrich for cellsexpressing B-cell receptors specific to this antigen. In anotherembodiment, the antigen is rendered multimeric, for example tetrameric,in order to increase the affinity of the cells expressing theappropriate B cell receptors. In another embodiment, these cells can belabeled using a fluorescently labeled antigen reagent. These cells canthen be enriched using a flow cytometry method that sorts based on thefluorescent label. This process can be done in combination with othermarkers of B-cells in the flow cytometry methodology. In anotherembodiment the fluorescent antigen reagent is rendered multimeric inorder to increase the affinity of the cells expressing the appropriate Bcell receptor.

One aspect of this invention is that the strength of the interaction ofthe different clonotypes with the specific antigen can be defined. Thedegree of enrichment of a clonotype by the antigen interaction providesa measure of the strength of the interaction. As a result instead of thetraditional “titer” level of an antibody, more detailed information canbe obtained. Specifically, the frequency of different clonotypes withdifferent avidities can be determined. In one embodiment, the antigen soused in enrichment is a single molecular species. In another embodiment,the antigen is a complex mixture of antigens that are relevant to adisease. The antigen may be a cell type or a mixture of cell types.

In another embodiment, the cells of interest are T cells that interactwith a specific antigen in the context of an MHC molecule. In oneembodiment, the peptide complexed with the MHC molecules is used tocapture the relevant cells. Tetramers of MHC-peptide complex have beenpreviously successfully used for this purpose. In another embodiment,blood or relevant tissue containing cells capable of antigenpresentation and T cells is incubated with the antigen to allow peptidesto be presented to the T cells. The cells that are activated throughbinding to these antigens can then be enriched by some feature ofactivation. Potentially any activation feature can be utilized like cellproliferation, leukocyte migration, cytotoxicity, and/or expression ofactivation markers. For example activated cells proliferate and they canbe allowed to divide and become enriched. Similarly, the activated cellscan express some markers that can be used to capture them. These markerscan be surface markers or some internal marker like cytokines, such asINFγ, IL-2, or IL-17. Cells expressing surface markers can be readilycaptured using different techniques such as FACS or beads coated withantibodies against the surface marker. Techniques to capture cellsexpressing intracellular markers, particularly cytokines have also beendeveloped. One technique is called Intracellular Cytokine Staining. Inthis method, cytokines specific to the immune process in question aretrapped within the T cells which are subsequently permeabilized allowingthese specific cytokines to be labeled using fluorescent antibodies.These labeled cells can then be enriched using a flow cytometry method.Another method, cytokine capture, uses hybrid antibodies that have dualspecificity. One of the specificities is to some generic marker in all Tcells (like MHC molecule) and the other is to the cytokine of interest,e.g., INFγ, IL-2, or Il-17. The generic specificity attaches theantibody on the surface of all T cells, and cytokines released from theT cells is then captured by antibodies attached on the same cells.Fluorescent antibodies against the relevant cytokine can then be usedallowing for the capture of the relevant cells using FACS.

One aspect of this invention is that the strength of the interaction ofthe different clonotypes with the specific antigen can be defined. Thedegree of enrichment of a clonotype by the antigen interaction providesa measure of the strength of the interaction. Therefore the frequency ofdifferent clonotypes with different avidities can be determined.

In one embodiment, the antigen so used in enrichment is a singlemolecular species. In another embodiment, the antigen is a complexmixture of antigens that are relevant to a disease. The complex mixtureof antigens can be a cell type or a mixture of cell types.

J. Antigen Enrichment for the Detection of Recurrence of LatentInfection

It is often of use in infectious disease to not only measure thepresence or absence of a pathogen but also to measure and monitor immuneresponse to this pathogen. As a result the measurement of antibodiesraised by the immune system against specific pathogen antigens is amethodology in routine clinical practice. Such immune responses tospecific pathogen antigen as measured by antibodies do not, however,give a comprehensive view of immune response to the antigen. Theantibodies measured, may be the product of many different B cell cloneseach of which is expressing a slightly different antibody each of whichmay carry slightly different information about the disease state.Furthermore, T-cell responses to these antigens are not being measuredat all.

The immune response of a patient to a pathogenic infection could be verycomprehensively profiled using the methods disclosed in this invention.In one embodiment, the B cell response can be comprehensively measuredin an individual infected with a pathogen using a B cell enrichment atone point in the disease course to ascertain the B cell clonotypes thatare relevant to the antibody response to that infection. In orderachieve this, B cells that are involved in an immune response to apathogen would be identified by performing an enrichment using antigenspresent in the pathogen in question. These antigens could be a singleantigen species, a set of distinct antigen species, or a complex mixtureof antigens from the pathogen including the entire cells from thepathogen. Such antigens are then immobilized to a solid surface orfluorescently labeled and enrichment is carried out using either a beadbased binding protocol, a column based binding protocol, or a flowcytometry method in the case where the antigens are fluorescentlylabeled. The cells from the patient are profiled before and afterenrichment by separating individual DNA or RNA molecules from the B cellreceptor in two dimensions and sequencing individual molecules to form aBCR clonotype profile. Clonotype sequences that show a significantfrequency shift between the two clonotype profiles are then candidatesto be clonotypes that are responsible for an immune response to theantigen(s). Further algorithms can optionally be developed to refine theprediction of which clonotypes are likely to be relevant to thisspecific immune response. These algorithms can use sequence parameterssuch as frequency, sequence length, amino acid sequence similarity,similarity to other similar clonotype sequences including those createdby somatic hypermutations, etc.

In a preferred embodiment, the antigen capture is done at onecalibration point in time to identify the relevant B cells and not doneand subsequent profiling time points in which all B cells are profiledwithout enrichment.

In another embodiment the T cell response is measured. In order achievethis, T cells that are involved in an immune response to a pathogenwould be identified by performing an enrichment using antigens presentin the pathogen in question. These antigens could be a single antigenspecies, a set of distinct antigen species, or a complex mixture ofantigens from the pathogen including the entire cells from the pathogen.In one embodiment, tetramers of MHC-antigen complex are used tofluorescently label the T cells. In another embodiment, such antigensare added to the blood of a patient at least a first time point andincubated so as to allow antigenic peptides to be presented by antigenpresenting cells of this individual. In both of these embodiments, inindividual spatially isolated RNA or DNA molecules from blood samplesare then profiled before and after the enrichment for these T cellsusing either tetramers of the MHC-antigen complex, the internal cytokinestaining method or the cytokine capture and FACS sorting. T cellclonotype sequences that show a significant frequency shift between thetwo clonotype profiles are then candidates to be clonotypes that areresponsible for an immune response to the antigen(s). Further algorithmscan optionally be developed to refine the prediction of which clonotypesare likely to be relevant to this specific immune response. Thesealgorithms can use sequence parameters such as frequency, sequencelength, amino acid sequence similarity, similarity to other similarclonotype sequences, etc.

In a preferred embodiment, the antigen capture is done at onecalibration point in time to identify the relevant B cells and not doneand subsequent profiling time points in which all B cells are profiledwithout enrichment.

IV. Determining Disease States

Because the immune system is so central to human health, the ability tomeasure immune responses has wide applications in medicine. Thisinvention teaches the ability to use the immune system to understandunderlying disease state when it is mediated by the immune system. Thisallows a very powerful set of diagnostic and prognostic applicationsthat use the immune profiles to inform the risks of wide variety ofclinical outcomes and allow physicians to intervene more effectively.

A. Utility of Immune Profiling in Autoimmune Disease Treatment

The methods of the provided invention can be used to diagnose and treatautoimmune disease in a subject. Autoimmune disease involves adaptiveimmune cells escaping the usual process conferring autoimmunity andattacking some target(s) on bodily tissue. Autoimmune diseases include,for example, acute disseminated encephalomyelitis, Addison's disease,ankylosing spondylitis, anti-phospholipid antibody syndrome, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease,Behçet's disease, bullous pemphigoid, Celiac disease, Chagas disease,Chronic obstructive pulmonary disease, dermatomyositis, diabetesmellitus type 1, Goodpasture's syndrome, Graves' disease, Guillain-Barrésyndrome, Hashimoto's thyroditis, Hidradenitis suppurativa, Idiopathicthrombocytopenic purpura, Interstitial cystitis, multiple sclerosis,myasthenia gravis, neuromyotonia, pemphigus vulgaris, pernicious anemia,polymyositis, primary biliary cirrhosis, rheumatoid arthritis,scleroderma, systemic lupus erythematosus, Sjögren's syndrome, andvasculitis syndromes. The stages of these autoimmune diseases can bediagnosed using the methods of the provided invention. Treatments can besuggested to a subject based on the stage of the autoimmune disease.

Clinical information regarding a subject with an autoimmune disease, orsuspected of having an autoimmune disease, can be used to determine thedisease state (or AutoImm load). Clinical information can be used toidentify patterns of a clonotype profile that correlate with a diseasestate. Clinical information can include, for example, height, weight,eye color, age, gender, ethnic group, blood pressure, LDL cholesterollevels, HDL cholesterol levels, family medical history, and molecularmarker information.

Clinical information can include symptoms of one or more autoimmunediseases. For autoimmune hepatitis symptoms can include fatigue,hepatomegaly, jaundice, pruritus, skin rash, arthralgia, abdominaldiscomfort, spider angiomas, nausea, vomiting, anorexia, dark urine,pale or gray stools. For dermatoymyositis (DM), symptoms can includerash (patchy, bluish-purple discolorations on the face, neck, shoulders,upper chest, elbows, knees, knuckles and back) accompanying or precedingmuscle weakness, dysphagia, myalgia, fatigue, weight loss and low-gradefever. For Graves' disease, symptoms can include weight loss due toincreased energy expenditure, increased appetite, heart rate and bloodpressure, and tremors, nervousness and sweating. For Hashimoto'sthyroiditis, symptoms can include mental and physical slowing, greatersensitivity to cold, weight gain, coarsening of the skin, goiter. Formixed connective tissue disease (MCTD)), symptoms can include featuresof systemic lupus erythematosus (SLE), scleroderma and polymyositis. ForPemphigoid, bullous (BP) symptoms can include mildly pruritic welts tosevere blisters and infection, oral or esophageal bullae. For pemphigus,symptoms can include blistering of skin and mucous membranes. Forpernicious anemia, symptoms can include shortness of breath, fatigue,pallor, tachycardia, inappetence, diarrhea, tingling and numbness ofhands and feet, sore mouth and unsteady gait. For polymyositis (PM),symptoms can include muscle weakness, dysphagia and myalgia. For primarybiliary cirrhosis (PBC), symptoms can include fatigue and pruritus. Forscleroderma (systemic sclerosis), symptoms can include swelling andpuffiness of the fingers or hands, skin thickening, skin ulcers on thefingers, joint stiffness in the hands, pain, sore throat and diarrhea.For Sjögren's syndrome, symptoms can include dryness of the eyes andmouth, swollen neck glands, difficulty swallowing or talking, unusualtastes or smells, thirst and tongue ulcers. For systemic lupuserythematosus (SLE)), symptoms can include fever, weight loss, hairloss, mouth and nose sores, malaise, fatigue, seizures and symptoms ofmental illness, joint inflammation similar to RA, butterfly rash on noseand cheeks, extreme sensitivity to cold in the hands and feet. Forvasculitis syndromes, e.g., Wegener's granulomatosis, idiopathiccrescentic glomerulonephritis (ICGN), microscopic polyarteritis (MPA),pulmonary renal syndrome (PRS), symptoms can include fatigue, weakness,fever, arthralgia, abdominal pain, renal problems and neurologicalproblems. The clinical information can be from one or more subjects atone or more points of time.

The clinical information can include information regarding responses ofa subject with an autoimmune disease to one or more treatments thesubject has received.

The clinical utility of AutoImm Load is discussed for specificautoimmune diseases below. Another embodiment of this inventioncontemplates the combination of the immune profiling tests with othermarkers that are already in use for the detection of disease activity inthese diseases to allow tests with greater sensitivity and specificity.Other molecular identifiers or markers can be used in computing theAutoImm Load or for determining the disease state. Molecular identifierscan include nucleic acids, proteins, carbohydrates, and lipids, andexpression profiles of nucleic acids or proteins. The molecularidentifiers can be of human or non-human origin (e.g., bacterial). Theidentifiers or markers can be determined by techniques that include, forexample, comparative genomic hybridization (CGH), chromosomal microarrayanalysis (CMA), expression profiling, DNA microarray, high-densityoligonucleotide microarray, whole-genome RNA expression array, peptidemicroarray, enzyme-linked immunosorbent assay (ELISA), genomesequencing, copy number (CNV) analysis, small nucleotide polymorphism(SNP) analysis, immunohistochemistry, in-situ hybridization, fluorescentin-situ hybridization (FISH), PCR, Western blotting, Southern blotting,SDS-PAGE, gel electrophoresis, and Northern blotting.

For systemic lupus erythematosus, markers can include levels oferythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels,Anti-ds DNA, other autoantibody titers, complement levels, urine proteinlevels, Urine protein/creatinine ratio, creatinine levels, blood ureanitrogen (BUN) levels, platelet levels, WBC counts, hematocrit (Hct),Hb, and urinalysis results. Other tests that are related for instance toSLE that can be integrated include, for example, CD27 level, CD27++ celllevel, INF-responsive genes, and chemokine score.

1. Systemic Lupus Erythematosus (SLE)

The methods of the provided invention can be used to determine states orstages of systemic lupus erythematosus (SLE or lupus). SLE is a seriousautoimmune condition that often afflicts young adults (mostly females).It is characterized by inflammatory processes that can affect manyorgans including the skin, joints, kidneys, lungs, heart, and centralnervous system leading to frequent disabilities and sometimes death. Thedisease follows a very unpredictable course marked by flare periodsfollowed by quiescent periods of remission. Nevertheless, patientsdiagnosed with SLE are seen regularly by a rheumatologist and treatedwith a variety of serious medications. These medications includesteroids such as Prednisone and other immunosuppressants such asCellcept (mycophenolate mofetil). While these drugs can reduce organdamage they contain significant side effects including risk of infectionand infertility. The unreliability for some of the symptoms (e.g., painand fatigue) and the unpredictable disease course makes tailoringmedication doses difficult, resulting in an overtreatment of somepatients and under-treatment of others. As a result, the treatment ofSLE poses significant therapeutic challenges to the clinician.

There are a number of standard methods a clinician can use to assess theactivity of SLE. The status of the disease can be measured by observingthe clinical symptoms of the disease. These methods include assessmentof signs (e.g., skin rash) and symptoms (e.g., joint pain and fatigue)as well as lab results (e.g., urine protein/creatinine ratio, anti-dsDNA antibody, and blood counts). These clinical markers, however, can belagging indicators of disease status and as such patients may respondonly after weeks or months of therapy. Furthermore, in some casessymptoms can be difficult to assess with precision (e.g., pain andfatigue). Other markers of inflammation, for example anti-ds DNAantibody, complement level (e.g., C3), C reactive protein (CRP), anderythrocyte sedimentation rate (ESR) usually lack specificity and/orsensitivity. Invasive methods such as kidney biopsy are impractical forroutine use. As a result clinicians perform quite a frequent testing oftheir patients without a perfect measure of the disease status. Theclinical symptoms and laboratory assessment are integrated in measuressuch as Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) andPhysician Global Assessment (PGA). These measures are not done routinelyin clinical practice and often fall short in several clinicalsituations.

In one embodiment of the invention clonotypes are identified whichrepresent different immune profiles for different disease states. Thedisease state is then tracked by comparing the identified immune profileto the patient's current immune profile. The disease can be lupus. Thedisease states can be flare periods and non-flare periods. This immuneprofile may be used as an early indicator of a flare state. This maydrive treatment decisions.

Specific examples of the utility of AutoImm Load in making therapeuticinterventions in SLE are discussed in greater detail in the examplessection along with specific enabling studies that determine AutoImmLoad.

In one aspect, SLE correlated clonotypes are related to antibodiesspecific for self antigens. Accordingly, a method of determining alikelihood that an individual has systemic lupus erythematosus comprisesthe following steps: (a) determining a profile of clonotypes from asample of B cells of the individual, the sample comprising a repertoireof clonotypes thereof; and (b) comparing clonotypes of the profile withclonotypes of an antigen-specific clonotype database to determine alevel of clonotype matches, thereby determining a likelihood of systemiclupus erythematosus, the antigen-specific clonotype database includingsubstantially all clonotypes of human immunoglobulin chains specific forthe one or more antigens selected from the group consisting of doublestranded DNA, malondialdehyde, 4-hydroxynonenal, superoxide dismutase,nitrotyrosine, cardiolipin, ribosomal P protein, phospholipid, coreprotein of small nuclear ribonuclearprotein (Smith antigen), histone, U1small nuclear ribonuclearprotein, type I topoisomerase, centromericproteins, SS-A ribonuclearprotein, SS-B ribonuclearprotein, andhistidine-tRNA ligase.

2. Multiple Sclerosis (MS)

The methods of the provided invention can also be used to determinestates or stages of Multiple Sclerosis (MS). MS is an autoimmune diseasethat affects the brain and spinal cord (central nervous system).Symptoms vary, because the location and severity of each attack can bedifferent. Episodes can last for days, weeks, or months. These episodesalternate with periods of reduced or no symptoms (remissions). It iscommon for the disease to return (relapse). However, the disease maycontinue to get worse without periods of remission.

Because nerves in any part of the brain or spinal cord may be damaged,patients with multiple sclerosis can have symptoms in many parts of thebody. Muscle symptoms include, for example, loss of balance, numbness orabnormal sensation in any area, pain because of muscle spasms, pain inthe arms or legs, problems moving arms or legs, problems walking,problems with coordination and making small movements, slurred ordifficult-to-understand speech, tremor in one or more arms or legs,uncontrollable spasm of muscle groups (muscle spasticity), and weaknessin one or more arms or legs.

Eye symptoms include, for example, double vision, eye discomfort,uncontrollable rapid eye movements, and vision loss (usually affects oneeye at a time).

Other brain and nerve symptoms include, for example, decreased attentionspan, decreased judgment, decreased memory, depression or feelings ofsadness, dizziness and balance problems, facial pain, hearing loss, andfatigue.

Bowel and bladder symptoms include, for example, constipation,difficulty beginning urinating, frequent need to urinate, stool leakage,strong urge to urinate, and urine leakage (incontinence).

There is no known cure for multiple sclerosis at this time. However,there are therapies that may slow the disease. The goal of treatment isto control symptoms and help the patient maintain a normal quality oflife.

Medications used to slow the progression of multiple sclerosis caninclude, for example, immune modulators to help control the immunesystem, including interferons (Avonex, Betaseron, or Rebif), monoclonalantibodies (Tysabri), glatiramer acetate (Copaxone), mitoxantrone(Novantrone), methotrexate, azathioprine (Imuran), cyclophosphamide(Cytoxan), and natalizumab (Tysabri). Steroids can be used to decreasethe severity of attacks.

Medications to control symptoms can include, for example, medicines toreduce muscle spasms such as Lioresal (Baclofen), tizanidine (Zanaflex),or a benzodiazepine, cholinergic medications to reduce urinary problems,antidepressants for mood or behavior symptoms, and amantadine forfatigue.

MS affects women more than men. The disorder most commonly beginsbetween ages 20 and 40, but can be seen at any age. MS is a progressivedisease, meaning the nerve damage (neurodegeneration) gets worse overtime. How quickly MS gets worse varies from person to person.Inflammation occurs when the body's own immune cells attack the nervoussystem. Repeated episodes of inflammation can occur along any area ofthe brain and spinal cord. People with a family history of MS and thosewho live in a geographical area with a higher incidence rate for MS havea higher risk of the disease.

Symptoms of MS may mimic those of many other nervous system disorders.The disease is diagnosed by ruling out other conditions. People who havea form of MS called relapsing-remitting may have a history of at leasttwo attacks, separated by a period of reduced or no symptoms. The healthcare provider may suspect MS if there are decreases in the function oftwo different parts of the central nervous system (such as abnormalreflexes) at two different times. A neurological exam may show reducednerve function in one area of the body, or spread over many parts of thebody.

Tests to diagnose multiple sclerosis include, for example, cerebrospinalfluid tests, including CSF oligoclonal banding, head MRI scan, lumbarpuncture (spinal tap), nerve function study (evoked potential test), andspine MRI.

Like other autoimmune diseases, MS follows an unpredictable course withacute flares and periods of remission. There are increasing numbers oftherapies, each with side effects that range from serious (weight gainand depression) to life threatening (pancytopenia and PML infections),variable effectiveness in different patients, and high costs. At thesame time, the lack of highly accurate and specific routine tests of MSdisease activity make the challenge of effectively administering therapycomplicated. Clinical episodes can be separated by long time periods (upto years in early stage disease) even without treatment. In addition,available medications reduce the likelihood of relapse but do notcompletely prevent them. Therefore disease activity is difficult toassess and thus, there is an inadequate short term measure of diseaseactivity that could be used to measure whether a specific therapy isshowing efficacy in a given patient by measuring the reduction in numberor severity of relapses. The only other test available for monitoringdisease activity is brain MRI to track the state of lesions as revealedwith the aid of contrast enhancing agents such as gadolinium. However,such imaging offers only an integrated view of brain damage and lacksspecificity and time resolution. Attempting to use MRI imaging to followdisease course on time scales shorter than a year is impractical giventhe costs, the lack of specificity and the dangers of excessive contrastexposure. As a result, patients are often treated at great expense forprolonged periods of time without any effective feedback that wouldallow the physician to modify dosing and/or switch of add therapies.

In one embodiment of the invention clonotypes are identified whichrepresent different immune profiles for different disease states. Thedisease state is then tracked by comparing the identified immune profileto the patients current immune profile. The disease can be MS. Thedisease states can be remission periods and active periods. This immuneprofile may be used as an early indicator of a remission ornon-remission period. This may drive treatment decisions.

3. Rheumatoid Arthritis (RA)

The methods can be used to measure disease status for Rheumatoidarthritis patients. Rheumatoid arthritis (RA) is a chronic, systemicinflammatory disorder that can affect many tissues and organs butprincipally attacks the joints, producing an inflammatory synovitis thatoften progresses to destruction of the articular cartilage and ankylosisof the joints. Rheumatoid arthritis can also produce diffuseinflammation in the lungs, pericardium, pleura, and sclera, and alsonodular lesions, most common in subcutaneous tissue under the skin.Although the cause of rheumatoid arthritis is unknown, autoimmunityplays a pivotal role in its chronicity and progression.

About 1% of the worlds population is afflicted by rheumatoid arthritis,women three times more often than men. Onset is most frequent betweenthe ages of 40 and 50, but people of any age can be affected. It can bea disabling and painful condition, which can lead to substantial loss offunctioning and mobility. RA is diagnosed chiefly on symptoms and signs,but can also be diagnosed with blood tests (especially a test calledrheumatoid factor) and X-rays. Diagnosis and long-term management aretypically performed by a rheumatologist, an expert in the diseases ofjoints and connective tissues.

Various treatments are available. Non-pharmacological treatment includesphysical therapy, orthoses, and occupational therapy. Analgesia(painkillers) and anti-inflammatory drugs, including steroids, can beused to suppress the symptoms, while disease-modifying antirheumaticdrugs (DMARDs) can be used to inhibit or halt the underlying immuneprocess and prevent long-term damage. In recent times, the newer groupof biologics has increased treatment options.

When RA is clinically suspected, immunological studies can be performed,such as testing for the presence of rheumatoid factor (RF, a specificantibody). A negative RF does not rule out RA; rather, the arthritis iscalled seronegative. This is the case in about 15% of patients. Duringthe first year of illness, rheumatoid factor is more likely to benegative with some individuals converting to seropositive status overtime. RF is also seen in other illnesses, for example Sjögren'ssyndrome, and in approximately 10% of the healthy population, thereforethe test is not very specific.

Because of this low specificity, new serological tests have beendeveloped, which test for the presence of so called anti-citrullinatedprotein antibodies (ACPAs). Like RF, these tests are positive in only aproportion (67%) of all RA cases, but are rarely positive if RA is notpresent, giving it a specificity of around 95%. As with RF, there isevidence for ACPAs being present in many cases even before onset ofclinical disease.

The most common tests for ACPAs are the anti-CCP (cyclic citrullinatedpeptide) test and the Anti-MCV assay (antibodies against mutatedcitrullinated Vimentin). Recently, a serological point-of-care test(POCT) for the early detection of RA has been developed. This assaycombines the detection of rheumatoid factor and anti-MCV for diagnosisof rheumatoid arthritis and shows a sensitivity of 72% and specificityof 99.7%.

Also, several other blood tests can be done to allow for other causes ofarthritis, such as lupus erythematosus. The erythrocyte sedimentationrate (ESR), C-reactive protein, full blood count, renal function, liverenzymes and other immunological tests (e.g., antinuclear antibody/ANA)are all performed at this stage. Elevated ferritin levels can revealhemochromatosis, a mimic RA, or be a sign of Still's disease, aseronegative, usually juvenile, variant of rheumatoid.

The term Disease modifying anti-rheumatic drug (DMARD) originally meanta drug that affects biological measures such as ESR and hemoglobin andautoantibody levels, but is now usually used to mean a drug that reducesthe rate of damage to bone and cartilage. DMARDs have been found both toproduce durable symptomatic remissions and to delay or halt progression.This is significant, as such damage is usually irreversible.Anti-inflammatories and analgesics improve pain and stiffness but do notprevent joint damage or slow the disease progression.

There is an increasing recognition among rheumatologists that permanentdamage to the joints occurs at a very early stage in the disease. In thepast it was common to start therapy with just an anti-inflammatory drug,and assess progression clinically and using X-rays. If there wasevidence that joint damage was starting to occur, then a more potentDMARD would be prescribed. Ultrasound and MRI are more sensitive methodsof imaging the joints and have demonstrated that joint damage occursmuch earlier and in more sufferers than was previously thought. Peoplewith normal X-rays will often have erosions detectable by ultrasoundthat X-ray could not demonstrate. The aim now is to treat before damageoccurs.

There may be other reasons why starting DMARDs early is beneficial topreventing structural joint damage. From the earliest stages of thedisease, the joints are infiltrated by cells of the immune system thatsignal to one another in ways that may involve a variety of positivefeedback loops (it has long been observed that a single corticosteroidinjection may abort synovitis in a particular joint for long periods).Interrupting this process as early as possible with an effective DMARD(such as methotrexate) appears to improve the outcome from the RA foryears afterwards. Delaying therapy for as little as a few months afterthe onset of symptoms can result in worse outcomes in the long term.There is therefore considerable interest in establishing the mosteffective therapy with early arthritis, when the patient is mostresponsive to therapy and have the most to gain.

Traditional small molecular mass drugs used to treat arthritis include,for example, chemically synthesized DMARDs: azathioprine, ciclosporin(cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine,leflunomide, methotrexate (MTX), minocycline, and sulfasalazine (SSZ).Cytotoxic drugs include Cyclophosphamide.

The most common adverse events relate to liver and bone marrow toxicity(MTX, SSZ, leflunomide, azathioprine, gold compounds, D-penicillamine),renal toxicity (cyclosporine A, parenteral gold salts, D-penicillamine),pneumonitis (MTX), allergic skin reactions (gold compounds, SSZ),autoimmunity (D-penicillamine, SSZ, minocycline) and infections(azathioprine, cyclosporine A). Hydroxychloroquine may cause oculartoxicity, although this is rare, and because hydroxychloroquine does notaffect the bone marrow or liver it is often considered to be the DMARDwith the least toxicity. Unfortunately hydroxychloroquine is not verypotent, and is usually insufficient to control symptoms on its own.

Biological agents (biologics) can be produced through geneticengineering, and include, for example, tumor necrosis factor alpha TNFα)blockers—etanercept (Enbrel), infliximab (Remicade), adalimumab(Humira), Interleukin 1 (IL-1) blockers—anakinra (Kineret), monoclonalantibodies against B cells—rituximab (Rituxan), T cell costimulationblocker—abatacept (Orencia), Interleukin 6 (IL-6) blockers—tocilizumab(an anti-IL-6 receptor antibody) (RoActemra, Actemra)

Anti-inflammatory agents include, for example, glucocorticoids,Non-steroidal anti-inflammatory drugs (NSAIDs, most also act asanalgesics). Analgesics include, for example, paracetamol (acetaminophenin US and Canada), opiates, diproqualone, and lidocaine topical.

The challenge of treating RA lies in the fact that the disease is a longterm chronic illness with that can result in challenging disability forwhich a large range of treatments exist each of which has significantdrawbacks. Many of the DMARDs subject the patients to significant sideeffects including increased risk for serious infections, cancer, or evenautoimmune disease. Furthermore, the biologically derived drugs are veryexpensive, and the patient can be subjected to frequent injections.

A doctor initiating therapy for a patient faces many possible options.It would be desirable to get rapid feedback once a patient startstherapy to understand whether the patient is responding to the therapythat is chosen before the clinical manifestation presents itself.Imaging is not sensitive and is expensive and many blood markers such asCRP lack sufficient sensitivity. A test that would allow the physicianto rapidly determine the state of the disease would allow him or her toadjust the therapy quickly to a more effective therapy, saving thepatient from additional joint damage and more effectively using theexpensive therapies available.

A patient that has not experienced any acute flares since beginningtreatment may in fact still be experiencing ongoing inflammatory damageto the joints that has not manifested itself clinically. A test thatwould allow the doctor to differentiate this state from the backgroundwould allow the therapy to be adjusted to try to bring the patientcloser to a state in which no ongoing joint damage is being experienced.

Specific examples of how AutoImm Load can be used in managing RApatients are described in further detail in the examples section of thisdocument.

In one embodiment of the invention clonotypes are identified whichrepresent different immune profiles for different disease states. Thedisease state is then tracked by comparing the identified immune profileto the patients current immune profile. The disease is RA. The diseasestates can be, but are not limited to periods of high inflammation and abaseline. These immune profiles are used to drive treatment decisions.

4. Ankylosing Spondylitis

The methods can be used to detect disease activity for Ankylosingspondylitis. Ankylosing spondylitis (AS, from Greek ankylos, bent;spondylos, vertebrae), previously known as Bechterew's disease,Bechterew syndrome, and Marie Strümpell disease, a form ofSpondyloarthritis, is a chronic, inflammatory arthritis and autoimmunedisease. It mainly affects joints in the spine and the sacroilium in thepelvis, causing eventual fusion of the spine. It is a member of thegroup of the spondyloarthropathies with a strong genetic predisposition.Complete fusion results in a complete rigidity of the spine, a conditionknown as bamboo spine.

The typical patient is a young male, aged 18-30, when symptoms of thedisease first appear, with chronic pain and stiffness in the lower partof the spine or sometimes the entire spine, often with pain referred toone or other buttock or the back of thigh from the sacroiliac joint. Menare affected more than women by a ratio about of 3:1, with the diseaseusually taking a more painful course in men than women. In 40% of cases,ankylosing spondylitis is associated with an inflammation of the eye(iridocyclitis and uveitis), causing redness, eye pain, vision loss,floaters and photophobia. Another common symptom is generalized fatigueand sometimes nausea. Less commonly aortitis, apical lung fibrosis andectasia of the sacral nerve root sheaths may occur. As with all theseronegative spondyloarthropathies, lifting of the nails (onycholysis)may occur

There is no direct test to diagnose AS. A clinical examination and X-raystudies of the spine, which show characteristic spinal changes andsacroiliitis, are the major diagnostic tools. A drawback of X-raydiagnosis is that signs and symptoms of AS have usually been establishedas long as 8-10 years prior to X-ray-evident changes occurring on aplain film X-ray, which means a delay of as long as 10 years beforeadequate therapies can be introduced. Options for earlier diagnosis aretomography and magnetic resonance imaging of the sacroiliac joints, butthe reliability of these tests is still unclear. The Schober's test is auseful clinical measure of flexion of the lumbar spine performed duringexamination.

During acute inflammatory periods, AS patients will sometimes show anincrease in the blood concentration of C-reactive protein (CRP) and anincrease in the erythrocyte sedimentation rate (ESR), but there are manywith AS whose CRP and ESR rates do not increase so normal CRP and ESRresults do not always correspond with the amount of inflammation aperson actually has. Sometimes people with AS have normal level results,yet are experiencing a significant amount of inflammation in theirbodies.

Ankylosing spondylitis (AS, from Greek ankylos, bent; spondylos,vertebrae), previously known as Bechterew's disease, Bechterew syndrome,and Marie Strümpell disease, a form of Spondyloarthritis, is a chronic,inflammatory arthritis and autoimmune disease. It mainly affects jointsin the spine and the sacroilium in the pelvis, causing eventual fusionof the spine.

It is a member of the group of the spondyloarthropathies with a stronggenetic predisposition. Complete fusion results in a complete rigidityof the spine, a condition known as bamboo spine.

There are three major types of medications used to treat ankylosingspondylitis.: 1) Anti-inflammatory drugs, which include NSAIDs such asibuprofen, phenylbutazone, indomethacin, naproxen and COX-2 inhibitors,which reduce inflammation and pain Opioid analgesics have also beenproven by clinical evidence to be very effective in alleviating the typeof chronic pain commonly experienced by those suffering from AS,especially in time-release formulations. 2) DMARDs such as ciclosporin,methotrexate, sulfasalazine, and corticosteroids, used to reduce theimmune system response through immunosuppression; 3) TNFα blockers(antagonists) such as etanercept, infliximab and adalimumab (also knownas biologics), are indicated for the treatment of and are effectiveimmunosuppressants in as in other autoimmune diseases;

TNFα blockers have been shown to be the most promising treatment,slowing the progress of AS in the majority of clinical cases, helpingmany patients receive a significant reduction, though not elimination,of their inflammation and pain. They have also been shown to be highlyeffective in treating not only the arthritis of the joints but also thespinal arthritis associated with AS. A drawback, besides the often highcost, is the fact that these drugs increase the risk of infections. Forthis reason, the protocol for any of the TNF-α blockers include a testfor tuberculosis (like Mantoux or Heat) before starting treatment. Incase of recurrent infections, even recurrent sore throats, the therapymay be suspended because of the involved immunosuppression. Patientstaking the TNF medications are advised to limit their exposure to otherswho are or may be carrying a virus (such as a cold or influenza) or whomay have a bacterial or fungal infection.

AS affects produces symptoms that are very common in the healthypopulations. For example, a patient presenting complaining of severeback pain need not be experiencing an AS flare but rather might justhave routine back pain. The physician is forced to make a decision aboutwhether to treat these symptoms with expensive drugs with potentiallysevere side effects without a very precise view into the state of thedisease. CRP and ESR do not provide a very precise view of the diseasestatus. At the same time the course of the untreated disease can resultin debilitating long term spinal damage. This state of affairs leads toa difficult clinical challenge and significant overtreatment is used.The availability of an objective measure that reflects disease activitycan be of great help in the management of AS patients.

In one embodiment of the invention clonotypes are identified whichrepresent different immune profiles for different disease states. Thedisease state is then tracked by comparing the identified immune profileto the patients current immune profile. The disease is AS. The diseasestates can be, but are not limited to periods of high inflammation and abaseline. These immune profiles are used to drive treatment decisions.

B. Utility of Immune Profiling in Cancer Detection

These methods can be used to measure cancer risk. Cancer has become theleading cause of death in the industrialized world. Therefore methods oftreatment of cancer are in great need. Many approaches for cancertreatment are being attempted including the development of new smallmolecule drugs as well as antibodies targeting the tumor.

One set of methods that has been proposed is immunotherapy. Tumorsurveillance is one of the functions of cells of the immune system.There are several categories of tumor antigens that are recognized bythe immune system. The first category is comprised of antigens that arenovel generated by somatic mutation (point mutation or a translocation)in the tumor. Another category consists of antigens from proteins thatare only expressed in male germ cells that do not express MHC molecules.The dysregulation of gene expression in many tumors may allow some ofthese antigens to be expressed. A third category includes antigens fromproteins only expressed in particular tissues. The fourth categorycomprises antigens that are significantly overexpressed in the tumortissue. Finally the fifth category includes antigens that result fromabnormal posttranslational modification.

One of the properties of tumors is their ability to escape effectiveelimination by the immune system. It is thought that new mutationsacquired in the tumor allow it to go from the equilibrium phase (wherethe tumor is not completely eliminated but its growth is held in check)to the escape phase where the tumor grows without effective control bythe immune system. There are many mechanisms that tumors employ toescape the immune system. These mechanisms include the lack of specificantigenic peptides, or the co-stimulatory molecules that can activate Tcells. Other mechanisms include the tumor secretion of factor thatinhibit T cells and the creation of a tumor-induced privileged site bycreating a physical barrier separating the tumor from lymphocytes.Inducing the immune system to better fight the tumor as a strategy fortreating cancer is being studied and tested in multiple ways. Oneapproach is the adoptive T cell therapy. This approach focuses onidentifying T cells that are targeting tumor antigens through isolationof cells that are infiltrating the tumor and/or reacting to a specifictumor antigen. These T cells can be grown in vitro in conditions thatenhance their effectiveness, like the use of IL-2 and/orantigen-presenting cells. The expanded cells are then infused back tothe patient blood. Another approach is to use of retrovirus containingtumor-specific TCR. These retrovirus can be infused in the patient inspecial cells that later secrete the retrovirus allowing it to infect Tcells that then start expressing the tumor-specific TCR. Finally acommon approach is the use of vaccination. The premise of this approachof therapy is that immunization of the patient with one or more of thetumor antigens will stimulate the immune system ability to fight thetumor. Immunization is often done with the use of an adjuvant likeBacille Calmette-Guerin (BCG). This approach has been successful inpreventing viral-induced cancer as evident by the ability to preventcervical cancers induced by HPV-16 and HPV-18. However this has beenless successful in the treatment of other tumors.

Much of the improvement in mortality because of cancer has come aboutdue to the availability of better early detection methods leading forinstance to reduced rates of mortality in breast cancer and cervicalcancers. The mutability of tumors makes their early treatment much moreeffective than when they are detected late. Traditionally, looking forcancer detection biomarkers usually involved looking for markers thatare highly expressed in the cancer and are at low level or absent in thenormal tissue. This has led to the identification of several tumormarkers, like PSA. One problem with early detection of in cancer is thatthe greatest value in for cancer detection occurs when detection ofbiomarker is most difficult, i.e., the tumor is very small. Therefore inorder to have an effective cancer detection biomarker that candistinguish patients with small tumors from those that do not, thereneeds to be a tremendous difference in expression between the tumor andthe normal tissue due to the large difference in size between the tumorand the normal tissue. Additionally the marker needs to “spill”efficiently to the blood or other body fluid to allow detection using anon-invasive technique.

This invention teaches a novel mechanism for cancer detection using theimmune cell response. In this view cancer detection is not achieved bythe detection of a marker produced by the tumor itself but by the immunesystem response to the tumor. Specifically the profile of TCR and/or BCRcan provide an insight on whether the body is mounting a response to atumor or not. This can ameliorate some of the issues with currentbiomarkers. First the immune response is an amplification signal thatcan be easier to detect. Second lymphocytes pass through the bloodregularly and hence the relevant biomarker may readily present anddetectable in peripheral blood than traditional tumor biomarker. Finallythe problem of “background” biomarker material generated by the normaltissue is greatly reduced. The great diversity of T and/or B cellsprovide a way to detect the relevant biomarker with high sensitivity andspecificity, particularly with the recent availability of highthroughput methods for DNA sequencing. The approach of using the immunesystem response to cancer to detect it leverages the foundations laid tothis field by the promise of immunotherapy. However the risk for the twoapplications is probably quite different. To use the immune response tocancer for its detection does not require that the specific clonotype beeffective in treating the tumor but rather that it is associated withthe immune response to the tumor.

Another embodiment of this invention contemplates the combination of theimmune profiling tests with other markers that are already in use forthe detection of cancer to allow tests with greater sensitivity andspecificity. Other molecular identifiers or markers can be used incomputing the Load algorithm or for determining the disease state.Molecular identifiers can include nucleic acids, proteins,carbohydrates, and lipids, and expression profiles of nucleic acids orproteins. The molecular identifiers can be of human or non-human origin(e.g., bacterial). The identifiers or markers can be determined bytechniques that include, for example, comparative genomic hybridization(CGH), chromosomal microarray analysis (CMA), expression profiling, DNAmicroarray, high-density oligonucleotide microarray, whole-genome RNAexpression array, peptide microarray, enzyme-linked immunosorbent assay(ELISA), genome sequencing, copy number (CNV) analysis, small nucleotidepolymorphism (SNP) analysis, immunohistochemistry, in-situhybridization, fluorescent in-situ hybridization (FISH), PCR, Westernblotting, Southern blotting, SDS-PAGE, gel electrophoresis, and Northernblotting.

C. Utility of Immune Profiling in Transplant Medicine

These methods can be used to detect immune rejection of transplantedorgans. Transplantation of organs have become an integral part ofmedicine with over 25,000 solid organ (kidney, liver, heart, pancreas,and lung) transplants and more than 15,000 bone marrow transplantsoccurring in the US per year. These are generally complicated proceduresdone at tertiary care centers. To minimize the risk of transplantrejection, patients are often placed on immunosuppression for extendedperiods of time subjecting them to the risk of cancer and infections.Furthermore many transplants are rejected either acutely or years afterthe transplantation. In spite of these issues organ transplant remainsan essential treatment modality as patients with organ failures have fewother alternatives.

Solid organ transplant rejection primarily occurs due to response of theadaptive immune system to the transplanted organ. This is due to thepresence of alloantigens in the graft that are recognized by the host'simmune system. The rejection can occur in three different phases. Thefirst is the hyperacute phase within minutes of the transplant wherepreformed antibodies mount a response to the graft. The second is theacute rejection that occurs in first weeks or months after thetransplant. The last is chronic rejection that can occur years after thetransplantation. Given these risks care has been taken to minimize theimmunogenic differences between the donor and recipient. For example therisk of the hyperacute reaction is greatly reduced when the donor andrecipient are matched for their ABO subtypes as well as tested for crossmatching (determining whether the recipient has antibodies that reactwith the leukocytes of the donor) Similarly careful matching for theMajor HistoCompatability (MHC) is done to reduce acute rejection.However given that MHC molecules are very polymorphic it is very hard tofind to identify a perfect match. Monozygotic twins have a perfect MHCmatching. Similarly 1/4 siblings are expected to have a perfect MHCmatch. Unrelated individuals that have the same detected alleles per theclinical test often have differences due to other polymorphic sites thatare not tested in routine clinical practice. However even with perfectMHC matching from siblings, there is still a significant risk ofrejection due to the existence of minor histocompatibility antigens, andindeed acute rejection is very common occurring to more than half of thegrafts.

One might imagine that more aggressive testing of the MHC locus as wellas identification and matching the minor histocompatibility antigenswould significantly improve the graft rejection and possibly survivalrates. While that might be true the limited numbers of available donororgans available makes this task impractical as more aggressive testingmay significantly delay the identification of an appropriate graft to beused for each patient. Therefore, much of the progress that has occurredin the transplantation field was in the use of immunosuppressive agentsto prevent and treat rejection. Currently many drugs are utilized forthis purpose including: Azathioprine, corticosteroids, Cyclosporine,Tacrolimus, Mycophenolate Acid, Sirolimus, Muromonab-CD3, MonoclonalAnti-CD25 Antibody, Monoclonal Anti-CD20 Antibody, and Calcineurininhibitors.

Bone marrow transplant is most frequently used for leukemia and lymphomatreatment. Typically the recipient undergoes an aggressive regimen ofradiation and/or chemotherapy to decrease the load of the tumor beforethe transplantation. Mature T cells from the donor can attack some ofthe host tissues in the inverse rejection that is called Graft VersusHost Disease (GVHD). This is often manifested by rash, diarrhea, andliver disease. Careful matching of MHC can ameliorate but not eliminatethis problem. One solution is the depletion of the donor bone marrow invitro of mature T cells that are ultimately responsible for GVHD. Oneproblem with this is that the same phenomenon that causes GVHD may beresponsible for some of the therapeutic effect of bone marrow transplantthrough the graft vs. leukemia effect where donor T cells attack theremaining cancer cells. In addition depletion of donor T cells canexpose to patient to the risk of being immunodificient. Therefore therisk and benefits have to be balanced when considering these approaches.Patients are therefore often treated with immunosuppressants to preventas well as treat GVHD.

Current management of bone marrow but even more so for solid organtransplantation rely heavily on the treatment with strongimmunosuppressive agents. However given that these drugs havesignificant risks they are used in a manner to balance risk and benefit.However given that the risk for a specific patient at a particular timeis not well understood patients are treated with the dose where risk andbenefits are balanced for the average patient. Tests that can predictfuture rejection events may potentially be very helpful in tailoringtreatment to the patients at the appropriate times they need them. Thismay result in reduction in the immunosuppressive doses or some of thepatients while improving the rate of rejection and hopefully graftsurvival.

Another embodiment of this invention contemplates the combination of theimmune profiling tests with other markers that are already in use forthe detection of transplant rejection to allow tests with greatersensitivity and specificity. Other molecular identifiers or markers canbe used in computing the Load algorithm or for determining the diseasestate. Molecular identifiers can include nucleic acids, proteins,carbohydrates, and lipids, and expression profiles of nucleic acids orproteins. The molecular identifiers can be of human or non-human origin(e.g., bacterial). The identifiers or markers can be determined bytechniques that include, for example, comparative genomic hybridization(CGH), chromosomal microarray analysis (CMA), expression profiling, DNAmicroarray, high-density oligonucleotide microarray, whole-genome RNAexpression array, peptide microarray, enzyme-linked immunosorbent assay(ELISA), genome sequencing, copy number (CNV) analysis, small nucleotidepolymorphism (SNP) analysis, immunohistochemistry, in-situhybridization, fluorescent in-situ hybridization (FISH), PCR, Westernblotting, Southern blotting, SDS-PAGE, gel electrophoresis, and Northernblotting.

D. Utility of Immune Profiling in the Treatment of Infection

These methods have utility in guiding the treatment of infectionsparticularly when these infections can exist in active and latentstates. The advent of antibiotics for the treatment of infectiousdisease over the past century has made a great impact on lifeexpectancy. Over the past decade molecular diagnostics techniques havetaken a rapidly increasing role in the diagnosis and management ofinfectious disease. The excellent sensitivity and specificity providedby nucleic acid amplification has enabled the application of thesetechniques to an increasing number of applications. Many of theapplications are used for the diagnostic evaluation of the presence orabsence of infectious agents. For example the testing of sexuallytransmitted diseases is often done by a molecular testing employingnucleic acid amplification technique. Another set of application involvethe assessment of the “load” of the infection in a patient with analready diagnosed infectious agent. An example of that is the assessmentof HIV viral load in patients already diagnosed with AIDS. This testhelps the physician in determining whether the state of the patient'sdisease and hence can provide guidance on the effectiveness of thetreatment regimen being used.

It is sometimes helpful not only to consider the level of the infectiousagent but also the immune response to the infectious agent. One examplewhere the immune response to the infection is used routinely in clinicalpractice is in hepatitis B. One aspect of hepatitis B testing relies ondetecting the infectious agent through detection of hepatitis B antigensof by a nucleic acid amplification assay. In addition it is common inroutine clinical practice to test for the presence of differentantibodies that target the hepatitis B virus. The presence of anti-HBcIgM usually occurs in an acute infection setting, the appearance ofanti-HBc IgG indicates the infection is chronic. Similarly the emergenceof anti-HBs antibody signals clearing of the infection.

In one embodiment of this invention the value of the assessing theimmune response to an infection is harnessed along with the sensitivityand specificity of the molecular testing. This can be particularlyuseful for infectious diseases that are chronic where the infectiousagent remains latent in the body. The profile of the TCR and/or BCR canbe used to assess the immune response to an infection. Sequencing can beused to obtain a profile of the TCR and/or BCR allowing the detection ofparticular clonotypes with high sensitivity and specificity. Todetermine the specific clonotypes that correlate with disease severalapproaches are conceived.

Another embodiment of this invention contemplates the combination of theimmune profiling tests with other markers that are already in use forthe detection of infectious agents to allow tests with greatersensitivity and specificity. Other molecular identifiers or markers canbe used in computing the Load algorithm or for determining the diseasestate. Molecular identifiers can include nucleic acids, proteins,carbohydrates, and lipids, and expression profiles of nucleic acids orproteins. The molecular identifiers can be of human or non-human origin(e.g., bacterial). The identifiers or markers can be determined bytechniques that include, for example, comparative genomic hybridization(CGH), chromosomal microarray analysis (CMA), expression profiling, DNAmicroarray, high-density oligonucleotide microarray, whole-genome RNAexpression array, peptide microarray, enzyme-linked immunosorbent assay(ELISA), genome sequencing, copy number (CNV) analysis, small nucleotidepolymorphism (SNP) analysis, immunohistochemistry, in-situhybridization, fluorescent in-situ hybridization (FISH), PCR, Westernblotting, Southern blotting, SDS-PAGE, gel electrophoresis, and Northernblotting.

E. Utility of Immune Profiling in the Treatment of Aging Patients

These methods have utility in monitoring the state of the immune systemin the aged. Older people suffer from a decline in the immune systemcalled immunosenescence that affects their ability to respond toinfections and to raise effective responses to vaccines (Weinberger etal., 2008). This is apparent from the high mortality rates due topneumonia in the elderly (Office for National Statistics, 2005), andtheir susceptibility to hospital-acquired infections, such asClostridium difficile and methicillin-resistant Staphylococcus aureus(Health Protection Agency, 2008). Furthermore the decline in the immunesystem ability is thought to explain the increased rate of cancers inthe elderly. In addition immunosenescence may contribute to other majordiseases of the elderly with significant component of inflammatoryprocesses, like Alzheimer and heart disease. An ability to predict whichindividuals are most at risk for these deadly outcomes would be usefulto geriatrics physicians as they make clinical decisions aboutvaccination, aggressive treatment of infections and hospitalization.

Many aspects of the innate and adaptive immune system are altered inimmunosenescence. T cells lose responsiveness, macrophages have adecreased antigen-presenting capacity and altered cytokine secretion,natural killer cells have reduced toxicity, follicular dendritic cellscannot present antigen as efficiently, and neutrophils lose phagocyticability. There is smaller pool of naïve T and B cells and an increase inthe memory and effector pool leading to a reduced diversity of T and Bcell repertoires leading to the reduction of the ability of the adaptiveimmune system to respond to new antigens. In particular T cellrepertoires that are associated with cytomegalovirus (CMV) are greatlyincreased and as much as 45% of the total T cell repertoire may bedevoted to it. It has been noted that these expansions are lesspronounced in centenarians.

Studies have suggested that immune markers can predict survival in theelderly. The degree of diversity of the B cell repertoire has been shownto predict survival in the elderly at least in one population. Eventhough these global differences in TCR and BCR diversity were shown topredict clinical outcomes but these markers lack specificity. Deeperanalysis of the repertoire data may provide significantly moreprediction accuracy. For example, expansions responsive to CMV may havea different significance than other expansions.

In one embodiment of this invention, RNA from the T and B cells found inperipheral blood can be collected from a longitudinal cohort of agingpatients whose clinical histories are followed for several years. TheTCRα and TCRβ genes and the IgH, IgK and IgL genes can be amplified ineach of these cohorts at several time points in their clinicalhistories. Profiles of patients with long survival is compared topatients with short survival. First, global measure of diversity can beobtained. This will include not only the number of different clonotypesidentified but also their diversity. For example, is the V, D, J segmentusage the same in the two groups or is one group more restricted in itsusage? For example two samples may have the same number of independentclonotype but the clonotypes for one of the two samples do not covermany of the V segments. It is logical to expect that this sample wouldbe less versatile in responding to a new antigen compared with the othersample whose clonotypes are distributed among all the V segments.

In addition to global diversity it is determined whether expandedclonotypes in patients who had a long survival can be distinguished onthe basis of some sequence parameter compared to clonotypes in patientswho had a short survival. This approach can be supplemented by lookingat clonotypes that respond to specific antigens. For example given theavailable evidence identification of CMV responsive clonotypes can havepredictive power. Capturing T cells clonotypes that are CMV reactive ina discovery study can be done from a set of elderly as well as healthypatients. Sequences of these clonotypes can be studied to identifyparameters that distinguish them from other clonotypes. Using thispredictive algorithm of CMV clonotypes with the longitudinal cohortdescribed above it can be assessed whether adding this information canadd to the ability to predict the patient who survive for a long timefrom that who does not.

Another embodiment of this invention contemplates the combination of theimmune profiling tests with other markers that are already in use forthe detection of health in the aging population to allow tests withgreater sensitivity and specificity. Other molecular identifiers ormarkers can be used in computing the Load algorithm or for determiningthe disease state. Molecular identifiers can include nucleic acids,proteins, carbohydrates, and lipids, and expression profiles of nucleicacids or proteins. The molecular identifiers can be of human ornon-human origin (e.g., bacterial). The identifiers or markers can bedetermined by techniques that include, for example, comparative genomichybridization (CGH), chromosomal microarray analysis (CMA), expressionprofiling, DNA microarray, high-density oligonucleotide microarray,whole-genome RNA expression array, peptide microarray, enzyme-linkedimmunosorbent assay (ELISA), genome sequencing, copy number (CNV)analysis, small nucleotide polymorphism (SNP) analysis,immunohistochemistry, in-situ hybridization, fluorescent in-situhybridization (FISH), PCR, Western blotting, Southern blotting,SDS-PAGE, gel electrophoresis, and Northern blotting.

F. Utility of Immune Profiling in the Administration of Vaccines

These methods have utility in the administration of vaccines. The use ofvaccination has led to a great reduction in the rate of infections ofmultiple organisms. One infectious disease that continues to have asignificant health impact with over 30,000 deaths a year in the US isinfluenza. Influenza vaccination has to be done yearly as the strainmutates rapidly. Most of the severe sequelae of the disease occur in theelderly. Unfortunately the elderly often experience immunosenescencerendering them inadequately responsive to the vaccination.

In order to distinguish patients who are responsive to vaccination fromthose that are not, a discovery study needs to be performed. In thispopulation pre and (at one or more set time) post vaccination bloodsamples are available for a cohort of Influenza vaccinated patients withknown Influenza outcome (i.e., were they later protected from theinfection or not). TCR and/or BCR sequence can be obtained from thesesamples. Clonotypes that are enriched after vaccination in each patientare determined. Enriched clonotypes in patients who responded to thevaccination are then compared to a control set of clonotypes (e.g., therest of the clonotypes in the same set of patients) to distinguish thecorrelating clonotypes from other clonotypes. The algorithm to predictthese clonotypes is then used to predict correlating clonotypes amongpatients who did not respond to the vaccination. Patients who did notrespond may generate the same type of clonotypes as those that respondedbut at lower levels. Alternatively it might be that non-respondersgenerate a distinct class of clonotypes. The number of correlatingclonotypes identified in the non-responder may distinguish these twopossibilities.

In another embodiment, an individual's responsiveness to a vaccinationis monitored by first obtaining a sample of lymphocytes from theindividual after vaccination from which lymphocytes reactive to thevaccine are isolated. Such isolation is readily carried out for B cellsusing conventional affinity purification with antigenic material from orrelated to the vaccine as capture moieties attached to a solid supportsuch as magnetic beads. Isolation of T cells may also be carried outwith conventional means, e.g. U.S. Pat. Nos. 7,776,562; 7,125,964;5,635,363; or the like, which are incorporated by reference. A clonotypeprofile is generated from the isolated sample of lymphocytes to obtain aset of correlating clonotypes. At subsequent time points, peripheralblood samples are obtained from the individual and clonotype profilesare generated. The rate of change of the frequency of correlatingclonotypes in the subsequent samples is monitored to determine theresponsiveness of the individual to the vaccination. Such method ofmonitoring responsiveness to a vaccination may be implemented with thefollowing steps: (a) enriching after a vaccination a sample oflymphocytes from peripheral blood of an individual to obtain a sample ofvaccine-responsive lymphocytes; (b) determining a clonotype profile fromthe sample of vaccine-responsive lymphocytes to identify one or morepatient-specific clonotypes correlated with vaccine response; and (c)determining a level of each of the one or more patient-specificclonotypes in a clonotype profile from a sample of peripheral bloodcells obtained at one or more subsequent times to monitor theresponsiveness of the individual to the vaccination. In one embodiment,responsiveness is determined by the increase in amount or frequency ofthe one or more patient-specific clonotypes in subsequently measuredclonotype profiles.

With the correlating clonotypes identified, an algorithm is then builtto generate a score for predicting likelihood of immunization. Data fromthe profiles of the vaccine-responders and those that do not respond areutilized to generate this algorithm. This algorithm can then be used topredict the likelihood of immunization in the next patient using thepredicted correlating clonotypes from a sample obtained afterimmunization. The prediction is done through the application of anotheralgorithm that has also been generated in the discovery study. It canoptionally be aided (or substituted) by data from the pre-calibration tolimit the search for correlating clonotypes to those that were enrichedafter immunization.

Another embodiment of this invention contemplates the combination of theimmune profiling tests with other markers that are already in use forthe detection of response to vaccination to allow tests with greatersensitivity and specificity. Other molecular identifiers or markers canbe used in computing the Load algorithm or for determining the diseasestate. Molecular identifiers can include nucleic acids, proteins,carbohydrates, and lipids, and expression profiles of nucleic acids orproteins. The molecular identifiers can be of human or non-human origin(e.g., bacterial). The identifiers or markers can be determined bytechniques that include, for example, comparative genomic hybridization(CGH), chromosomal microarray analysis (CMA), expression profiling, DNAmicroarray, high-density oligonucleotide microarray, whole-genome RNAexpression array, peptide microarray, enzyme-linked immunosorbent assay(ELISA), genome sequencing, copy number (CNV) analysis, small nucleotidepolymorphism (SNP) analysis, immunohistochemistry, in-situhybridization, fluorescent in-situ hybridization (FISH), PCR, Westernblotting, Southern blotting, SDS-PAGE, gel electrophoresis, and Northernblotting.

G. Utility of Immune Profiling in the Monitoring of ImmuneHypersensitivity (Allergy)

The adaptive immune system has evolved to respond to antigens that areassociated with pathogens. As in the case of autoimmune diseases, theimmune system can sometimes have the wrong target. Whereas in autoimmunediseases the immune system targets self antigen, in hypersensitivityreactions it mounts a response to harmless stimuli like medications,dust, and food. Hypersensitivity is very common with as many as 50% ofthe US population having allergy to an environmental stimulus, and it iscaused by mechanisms. Hypersensitivity is divided into 4 types. Type Ihypersensitivity is the immediate type hypersensitivity and is mediatedby IgE. Type II is often due to IgG antibody binding to cellsurface-associated antigen. For example a harmless drug that binds tothe surface of the cell can make the cell a target for anti-drug IgG inpatients who happened to have these antibodies. Type III is caused bydeposition of antigen-antibody complexes on tissues. This occurs forexample when the amount of antigen is large resulting in small immunecomplexes that can't be cleared efficiently and are instead deposited onblood vessel walls. Type IV sensitivity is a delayed typehypersensitivity mediated by T cells. Type I and type W have the highestimpact on human health.

In Type I hypersensitivity reaction the patient becomes sensitized to aharmless antigen (allergen) by producing IgE antibody against it. Laterexposure to the allergen induces the activation of IgE-binding cells,such as mast cells and basophils. Once activated these cells cause theallergic reaction through inducing an inflammatory process by secretingstored chemicals and synthesizing cytokines, leukotrienes, andprostaglandins. The dose and the route of entry of the allergendetermines the magnitude of the allergic reaction that can range fromsymptoms of allergic rhinitis to the life-threatening circulatorycollapse in anaphylaxis. Often the acute Type I reaction is laterfollowed by another late phase that is plays a role in many of theresulting pathological processes. The late phase of recruitment of Thelper cells and other inflammatory cells is essentially a Type IVhypersensitivity reaction. Some Type I allergic reactions includeseasonal rhinoconjunctivitis (hayfever), food allergy, drug-inducedanaphylaxis, atopic dermatitis (eczema), and asthma. These are verycommon conditions with rising prevalence causing significant costs aswell as morbidity and mortality. For example, Asthma is a chronicdisease that inflicts ˜7% of the US population causing ˜4,000 deaths ayear. Some of these diseases have some related aspects. For example,patients with atopic dermatitis are at significantly increased risk tohave asthma. Food allergy can cause vomiting and diarrhea but can alsoresult in anaphylaxis in a significant number of patients—30,000 casesresulting in ˜200 deaths per year in the US. Some of the same allergensthat activate submucosal mast cells in the nose causing symptoms ofallergic rhinitis can also activate mast cells in the lower airwayscausing bronchial constriction, a typical symptom of asthma. Some TypeIV hypersensitivity reactions are contact dermatitis (e.g., poison ivy),chronic rhinitis, chronic asthma, and celiac disease. Celiac disease isa chronic disease caused by a non-IgE mediated food allergy. It is adisease of the small intestine caused by the allergic response againstgluten, a component present in wheat and other foods. Over 95% ofpatients celiac patients have a specific MHC class II allele, theHLA-DQ2.

Treatment of hypersensitivity reactions differs, but they often had twoaspects: the acute treatment and chronic management or prevention. Someof these conditions can be life threatening (anaphylaxis, and acuteasthma) and involve immediate medical attention. The chronic managementin general it involves trying to avoid the specific allergen. This maybe effective when the allergen can be clearly identified (e.g., allergyto nuts), but this can be difficult when the allergen is present widelyin the environment, like pollen or dust. Therefore chronic treatmentwith medications is often utilized for some of these diseases (e.g.,asthma and allergic rhinitis). The level of effectiveness of thetreatment management is ultimately tested when the patient is re-exposedto the allergen(s). Therefore some patients may be subject to over- orunder-treatment. Ideally a test that assesses the disease activity andthe degree to which the patient is prone to mount a hypersensitivityresponse would be available. Such a test would allow the tailoring oftreatment to the individual patient needs.

H. Detection of Lymphoid Neoplasms

One aspect of this invention will utilize next generation sequencingtechnologies to evaluate the level of specific TCR or BCR rearrangementsin cancers of the lymphocytes. These sequencing technologies can obtainsequence reads from 1 million or more individually spatially isolatedTCR or BCR molecules at a reasonable cost. A sequence present at1/1,000,000 or lower can still be detected in a specific manner usingthese technologies thus allowing a cancer cell associated with aparticular TCR or BCR rearrangement to be detected at that level.Multiplex amplification to amplify all the different types of sequencesfor a specific gene can be done from blood or bone marrow DNA. Forexample to amplify IgH sequences, several primers complementary to allthe known V segments and alleles can be used along with several primerscomplementary to all the J segments and alleles. It is important thatlittle amplification bias occurs among the different sequences. We haveshown that we can amplify from RNA the TCRβ and IgH genes with onlysmall differences in the efficiencies of the different V primers, andthereby validating the possibility of doing the same from DNA which willallow the assessment of cancer cells even when the TCR or BCR is notbeing expressed.

The sensitivity this invention is determined by the counting statisticsof the individual spatially isolated molecules taking into account anybias in amplification of different clonotypes. Therefore it isanticipated that this approach will offer more sensitivity and will beless subject to sensitivity differences for different tumor cell TCR orBCR sequences when compared to real time PCR assays developed for thesame purpose. Furthermore to obtain more sensitivity one can simplyobtain more sequencing reads. Since the sequencing costs continue todrop we anticipate the sensitivity at a given cost will continue toimprove. With sufficient sequencing reads the sensitivity becomeslimited by the number of lymphocytes in the sample. In contrast,sensitivity for the real time PCR assay is limited by backgrounds causedby nonspecific amplifications and hybridizations of any probes which canbe substantial.

In order to use this invention to monitor lymphoid cancers a patient'sspecific clones can be determined by sequencing a diagnostic leukemia orlymphoma sample; that is, patient-specific clonotypes are determined bygenerating a clonotype profile from a sample from a disease-relatedtissue, where disease responsive lymphocytes are found in an enrichedstate. Once the clonotype profile is determined, levels ofdisease-related clonotypes can be determined by comparison to aclonotype profile of a sample from a tissue that is not associated withthe disease. Afterwards, levels of disease-related clonotypes aredetermined in clonotype profiles from samples from that patient atsubsequent time points in the disease course. Preferably, suchsubsequent samples are taken from tissues that are convenientlyaccessed, such as peripheral blood. The cells in the blood sample can beused and alternatively, DNA or RNA from the cell free plasma can beused. There is no requirement for a patient-specific probes or theutilization of patient-specific templates to be run as standards, as iscalled for in current technology. In this embodiment of the inventionpatient-specific clonotypes that are identified can be followed byobtaining full sequence repertoires and by informatically measuring therelevant correlating clonotypes based on matching the sequences obtainedto stored data regarding the relevant sequences for each patient.Disease-related tissues for lymphomas may include lymphoid tissues, bonemarrow, peripheral blood, and the like.

Identification of cancer clonotypes. In order to use the sequencingmethods to monitor cancer, it is critical to define the cancerclonotypes in each individual. For secondary testing (recurrence andprognosis application for patients diagnosed with lymphoid neoplasms)identification of the cancer clonotype can often be quitestraightforward. For example, blood or bone marrow samples of leukemiapatients at diagnosis typically exhibit the cancer clonotype as the mostfrequent clonotype in the sample. In other cases of secondary testing(for example biopsy from some lymphoma samples) the cancer clone may notbe present at very high levels. Other clonotypes that are reactive to avariety of antigens including those that are attacking the cancer mayhave tumor are found at higher frequency. If the level of the clonotypeis not by itself sufficient to determine the cancer clonotype othercriteria can be used. Several methods described below can be used toidentify the cancer clonotypes.

Cross lineage rearrangement. Some types of otherwise uncommonrearrangements are common in some cancers and thus can be used toassociate them with tumor. For example, cross lineage rearrangements,like T cell receptor (α β γ and/or δ) in B cells or B cell receptor(IgH, IgK, and/or IgL) in T cells are common, especially in ALL. Thepresence of cross lineage rearrangements is likely to support amalignant origin of the clonotype.

Sequencing cross lineage rearrangement using sequencing of specific celltypes. Alternatively, cells of one type can be isolated through standardmethods (e.g, magnetic beads and FACS) that utilize the presence ofcertain antigens on the surface of cells. Sequencing can be done toassess for the presence of cross lineage rearrangement. For example, Bcells can be isolated and sequencing TRCβ can be performed. The presenceof an enriched specific TCRβ sequence would be consistent with cancer.The sequencing can be performed before and after the enrichment allowingfor the determination of the level without enrichment and the degree ofenrichment.

Inactive immune receptor. Another feature that may be useful todistinguish malignant cells from others is that the non-malignant cellsneed to have an active immune cell receptor. Lymphocytes proliferate inreaction to antigens and may reach high levels. Therefore reaching highlevels for a normal (non-cancer) clonotype is dependent on an activeimmune receptor. Identification in a biopsy of a high levelnon-functional sequence is not sufficient to identify cancer sincenon-functional rearrangements can be found in the same cell as afunctional ones due to nonfunctional rearrangements of the second allelewithin a cell that contains a functional rearrangement. The use of RNAmay allow the disambiguation of this point as tumor cells may continueto express nonfunctional rearrangements while this is unlikely to occurin normal cells but in general more definitive methods are useful. Thereare additional features that may distinguish non-functional sequences incancer. For example, some of the immature cancers, e.g. ALL, often haveonly the IgH rearranged but not IgK or IgL. This pattern is not likelyto achieve high frequency in the absence of cancer. These nonfunctionalrearrangements can be assessed using the following techniques.

Statistical linkage in a series of diluted samples. An alternative tothe physical linking is to look for mapping linkage as described above.In this case only one gene (e.g, IgH) is being testing and the questionis what are the non-functional sequences linked to, i.e., what is thesequence of the other allele of IgH in the same cell. High frequencynon-functional alleles linked to a second nonfunctional allele areconsistent with achieving high frequency due to cancer.

Sequencing specific cell type. This pattern can also be identified bycapturing cells carrying one marker and assessing the sequence of theother. For example, one can use FCM to capture cells that are IgK andIgL negative. Sequencing IgH before and after the FCM enrichment canidentify clones that are enriched in this population. Cells that are IgKand IgL negative are not expected to reach high frequency and theirpresence is consistent with an immature cancer, like ALL.

Inactivating somatic hypermutations. An alternative pattern may be foundin B cell lymphomas whose clonotypes have undergone somatichypermutations. Some of these clones can have inter-clonal variability,in which the cancer cells comprise several clones with differentmutations within them. Some of the resulting clones may possibly haveinactivating mutations. For a normal antigen-driven somatichypermutation it is unlikely that a clone with an inactivating somatichypermutation is selected and expanded. The presence of such clones isconsistent with cancer.

Sequencing cell fractions carrying cancer markers. In cases where cancermarkers are known, FCM can be performed to enrich for cancer cells usingthis marker. Sequencing the immune receptor repertoire can be performedbefore and after the enrichment. Clonotypes that get enriched are likelyassociated with the cancer clonotype. For example, the lymphoma cellscan be enriched using FACS to isolate cells carrying the particularmarkers (most conveniently surface markers) relevant to the tumor.Sequencing BCR before and after enrichment would readily identify theenriched clonotype and by extension the cancer clonotype.

Alternatively the association of a marker can be assessed at the DNA orRNA level. This can be accomplished by several means including linkedPCR or statistical association with serially diluted cells as describedabove. Quantitating the linkage of these markers will enable betterperformance of the assay as many markers are overexpressed in cancercells but still present at some level in normal cells. To account forthis the linked PCR can be done using three genes: the immune receptor,the cancer marker, and the control gene. The immune receptor gene canlink with either of the other two genes and the fraction of linkedmolecules that are the result of a linkage between the receptorrearrangement and the cancer marker can indicate the level of expressionof this cancer marker.

Detection of translocations. In addition to serving as a marker of cellsthat have become cancerous IgH is often one of the two pathologicaltranslocation partners in lymphoid neoplasms. One example is thet(11:14) that puts the J segment of IgH in close proximity to thecycline D 1 (CCND1) gene resulting in its overexpression. Thisrearrangement which is referred to as BCL1-IgH occurs in as many as60-70% of mantle cell lymphoma as well as other lymphoid neoplasms (e.g,20% of multiple myeloma). Another example is t(14:18) that puts the Jsegment of IgH in close proximity to BCL2 resulting in its overexpression. This rearrangement occurs in up to 90% of follicularlymphoma and 20% of large B cell lymphoma. These rearrangements aretypically identified by cytogenetics, Southern blotting, or FISH. PCRhas the potential to identify rearrangement at very high sensitivity andspecificity as shown by BCR-ABL for the detection of Philadelphiachromosome. Different PCR techniques have been used to the assessment oftranslocations relevant to lymphoma, with the recently introduced realtime PCR (e.g, for BCL2-IgH) being probably the most advanced. There area few features of BCL1-IgH and BCL2-IgH that make their detection lesssensitive and specific than that of BCR-ABL. First, in contrast toBCR-ABL, BCL1-IgH and BCL2-IgH do not generate a fusion protein, andthere is no splicing event that generates predictable molecularstructure. Instead the breakpoints may span a large region. There arecommon breakpoints that allow the detection of up to 88% of BCL2-IgHusing a combination of primers and ˜40% of the BCL1-IgH. This results inmissing some patients that have the translocation. Second, theserearrangements may be present in normal individuals that would never getcancer. For example, BLC2-IgH translocation has been found at the levelof ˜10⁻⁵ in a large fraction of the normal individuals with over ˜4%carrying BCL2-IgH at a frequency of > 1/25K. The frequency of BCL2-IgHgets higher with increasing age. It is also hypothesized that differentpeople may have distinct levels of “background” translocation.Presumably the presence of this translocation in normal sample is due tothe fact that tumorgenesis is a multi-step process and the BCL2-IgH isnot sufficient for tumors to emerge. The presence of this low levelbackground puts a limit on the sensitivity of detection.

Amplification of with a pool of the J primers complementary to all the Jsegments and primers complementary to the regions upstream of the BCL1or BCL2 translocation breakpoints can be sequenced. This can generate amethod for sensitive detection of these translocations and the cancercells they appear in. First, deep sequencing of individual isolatedmolecules (e.g, 100K or 1 million reads) can allow the detection of theappropriate sequences from a small number of cells in a background ofamplifications of other loci. In addition, the problem of the backgroundtranslocations in normal individuals may ameliorate the problem thatreal time PCR suffer from. There is evidence that, at least in somecases, the background translocations are not clonal but rather appearrepeatedly in the same patient. Using sequencing one can distinguish thedifferent translocation events to obtain frequency of the independenttranslocation events. Since the breakpoint of different translocationsis likely to be distinct translocation events can be distinguished fromeach other. Alternatively or additionally, a linking PCR using thetranslocation with a B or T cell receptor gene can be done to provide aunique barcode for the translocation. The linking can also be donestatistically using a set of dilution samples as described above.

Serial monitoring of the level of the translocation to detect points atwhich they increase in frequency may be helpful in early cancerdetection as well as for detection of recurrence. In that latter casethe specific breakpoint relevant to the patient may be identified fromthe diagnostic biopsy. This concept of distinguishing translocations bytheir breakpoints through sequencing and therefore distinguishingbackground from cancer can be extended to other translocation thatinvolve IgH (e.g, t(8:14)) or all other translocations in lymphoidneoplasm or other cancers.

Changing Cancer cell levels and the likelihood of cancer. The merepresence of sequences that point to the existence of remaining tumorcell may not by itself predict a clinical relapse. For example a steadystate of tumor level may be achieved by the balancing forces of tumorcell proliferation and immune response to the tumor. It is anticipatedthat in addition to the absolute level of a clonotype, its rate ofchange can be informative in predicting the likelihood of relapse. Forexample consider two patients each with level X of their respectivepatient-specific cancer clonotypes. If the level on previous tests forone of the patients has been consistently X and the level for the otherpatient in previous tests has been considerably lower than thelikelihood of the second patient developing a relapse might be higherthan for the first patient.

Similarly additional data relating to the status of the cell containingthe cancer-related clonotype can be used to predict likelihood ofrecurrence. For example, the presence of certain markers (surface ornon-surface) can be an indication of the functional status of the celland hence the likelihood of recurrence. Sequencing before and after thecapture of cells with the relevant markers can determine the fraction ofcells with the cancer clonotype that carry the relevant markers.Similarly some markers relevant to the likelihood of recurrence (e.g.,expression of some gene relating to cell growth) can be assessed at theRNA level. This can be done by several methods including linking PCR orstatistically by cell dilution as described above. Finally, it ispossible that the level of immune receptor specific RNA in the tumorcell can have functional consequence and association with the likelihoodof recurrence. This level can be assessed by doing linking PCR between acontrol gene 1 that can link to either the immune receptor rearrangementor control gene 2. The relative fraction of the two products can beindicative of the relative amount of the RNA in the cell. Another methodinvolves comparing the RNA level to the DNA level of the immune receptorrearrangement. The frequency of the cancer-specific clonotype in the DNAidentifies the relative level of the cancer-specific clonotype. Thefrequency of the same clonotype can then be assessed from RNA, and therelative frequency in RNA and in DNA can be followed. A change in thisrelative frequency can be indicative of a change in the likelihood ofrecurrence.

Immune reaction to lymphoid cancer. In addition to monitoring the cancerclonotype and its potential progeny, we can also assess the immuneresponse to the tumor. We can identify clonotypes that are likelymounting a response against the tumor. For example B or T cellclonotypes that are enriched in the biopsy of the diagnostic lymph nodebiopsy may be the result of immune response to the tumor. Additionallyfunctional testing to identify T cells interacting with some tumorantigen can be done. This can be specific antigens or it can be thetumor cells themselves. For example sequencing of the TCR before andafter stimulation with antigen or tumor cells can identify the relevantT cells by the virtue of their enrichment after stimulation. The levelof these T cells in subsequent blood samples from the patient can behelpful in predicting relapse. For example consider the case mentionedabove where a specific level X of the cancer clonotype has been stablydetected. This is likely the result of balancing the tumor growth withthe immune response to the tumor. If at some point the immune responseto the tumor is reduced it is possible to anticipate that the tumor willrelapse. The immune response to the tumor can be quantitate throughdetermination of the level of the T cell clonotypes that have beendetermined to be capable of attacking the tumor.

Integration of sequencing with other cell markers. Detection of thecancer-specific clonotype can be done through sequencing of immunereceptor rearrangements as described above. Presence of markers (surfaceor non-surface) that are relevant to cancer cells can be used to capturecell subpopulations that are later sequenced. The combination of usingmarker-specific capture and sequencing before and after the capture canprovide additional information. First, this can be used to identify theclonotypes that are enriched and hence are likely to be thecancer-specific clonotypes as described above. In addition, sequencingcells that have cancer-specific markers can lead to highersensitivities. With a perfect marker, only few reads need to be done todetect cancer-specific clonotypes in millions of other cells. Mostmarkers do not perform perfectly and significant background (i.e.,non-cancer cells) is generated by their capture. However the enrichmentof cancer cells by these markers can lead either to equivalentsensitivity with less sequencing reads or to better sensitivity thandoing the same number of sequencing reads without enrichment. Forexample, with 1 million sequencing reads one can assess ˜1 million cellsthat are captured with a cancer-specific marker. This corresponds tomore cells that were present before capture and hence bettersensitivity. Finally the use of markers can provide functional aspectsthat relate to the tumor biology and prognosis. Some level of tumorcells may be present in the blood of different samples, but thefunctional marker on the cells may distinguish samples that indicatehigh likelihood of recurrence from those that predict low likelihood ofrecurrence. For example, samples can be sequenced before and aftercapture with a relevant marker and the percentage of the clonotypesequences with the specific marker can be assessed. Two samples with thesame total level of cancer-specific clonotypes but different fractionsof those cells carrying the relevant marker may be predicted to havedifferent likelihood of recurrence.

Clone Evolution and Detection of Phylogenetic Clones (Clans) and OtherCancer-Related Mutations

As mentioned above, in one aspect, methods of the invention monitor alevel of a clan of clonotypes rather than an individual clonotype. Thisis because of the phenomena of clonal evolution, e.g. Campbell et al,Proc. Natl. Acad. Sci., 105: 13081-13086 (2008); Gerlinger et al, Br. J.Cancer, 103: 1139-1143 (2010). The sequence of a clone that is presentin the diagnostic sample may not remain exactly the same as the one in alater sample, such as one taken upon a relapse of disease. Therefore ifone is following the exact clonotype sequence that matches thediagnostic sample sequence, the detection of a relapse might fail. Suchevolved clone are readily detected and identified by sequencing. Forexample many of the evolved clones emerge by V region replacement(called VH replacement). These types of evolved clones are missed byreal time PCR techniques since the primers target the wrong V segment.However given that the D-J junction stays intact in the evolved clone,it can be detected and identified in this invention using the sequencingof individual spatially isolated molecules. Furthermore, the presence ofthese related clonotypes at appreciable frequency in the diagnosticsample increases the likelihood of the relevance of the clonotype.Similarly the development of somatic hypermutations in the immunereceptor sequence may interfere with the real time PCR probe detection,but appropriate algorithms applied to the sequencing readout (asdisclosed above) can still recognize a clonotype as an evolvingclonotype. For example, somatic hypermutations in the V or J segmentscan be recognized. This is done by mapping the clonotypes to the closestgerm line V and J sequences. Differences from the germ line sequencescan be attributed to somatic hypermutations. Therefore clonotypes thatevolve through somatic hypermutations in the V or J segments can bereadily detected and identified. Somatic hypermutations in the NDNregion can be predicted. When the remaining D segment is long enough tobe recognized and mapped, any somatic mutation in it can be readilyrecognized. Somatic hypermutations in the N+P bases (or in D segmentthat is not mappable) cannot be recognized for certain as thesesequences can be modified in newly recombined cells which may not beprogeny of the cancerous clonotype. However algorithms are readilyconstructed to identify base changes that have a high likelihood ofbeing due to somatic mutation. For example a clonotype with the same Vand J segments and 1 base difference in the NDN region from the originalclone(s) has a high likelihood of being the result of somaticrecombination. This likelihood can be increased if there are othersomatic hypermutations in the V and J segments because this identifiesthis specific clonotype as one that has been the subject of somatichypermutation. Therefore the likelihood of a clonotype being the resultof somatic hypermutation from an original clonotype can be computedusing several parameters: the number of differences in the NDN region,the length of NDN region, as well as the presence of other somatichypermutations in the V and/or J segments.

The clonal evolution data can be informative. For example if the majorclone is an evolved clone (one that was absent previously, andtherefore, previously unrecorded) then this is an indication of thattumor has acquired new genetic changes with potential selectiveadvantages. This is not to say that the specific changes in the immunecell receptor are the cause of the selective advantage but rather thatthey may represent a marker for it. Tumors whose clonotypes have evolvedcan potentially be associated with differential prognosis. In one aspectof the invention, a clonotype or clonotypes being used as apatient-specific biomarker of a disease, such as a lymphoid neoplasm,for example, a leukemia, includes previously unrecorded clonotypes thatare somatic mutants of the clonotype or clonotypes being monitored. Inanother aspect, whenever any previously unrecorded clonotype is at leastninety percent homologous to an existing clonotype or group ofclonotypes serving as patient-specific biomarkers, then such homologousclonotype is included with or in the group of clonotypes being monitoredgoing forward. That is, if one or more patient-specific clonotypes areidentified in a lymphoid neoplasm and used to periodically monitor thedisease (for example, by making measurement on less invasively acquiredblood samples) and if in the course of one such measurement a new(previously unrecorded) clonotype is detected that is a somatic mutationof a clonotype of the current set, then it is added to the set ofpatient-specific clonotypes that are monitored for subsequentmeasurements. In one embodiment, if such previously unrecorded clonotypeis at least ninety percent homologous with a member of the current set,then it is added to the patient-specific set of clonotype biomarkers forthe next test carried out on the patient; that is, the such previouslyunrecorded clonotype is included in the clan of the member of thecurrent set of clonotypes from which it was derived (based on the aboveanalysis of the clonotype data). In another embodiment, such inclusionis carried out if the previously unrecorded clonotype is at leastninety-five percent homologous with a member of the current set. Inanother embodiment, such inclusion is carried out if the previouslyunrecorded clonotype is at least ninety-eight percent homologous with amember of the current set.

It is also possible that a cell evolves through a process that replacesthe NDN region but preserves the V and V segment along with theiraccumulated mutations. Such cells can be identified as previouslyunrecorded cancer clonotypes by the identification of the common V and Jsegment provided their contain a sufficient number of mutations torender the chance of these mutations being independently derived small.A further constraint may be that the NDN region is of similar size tothe previously sequenced clone.

Assessment of Sufficient Number of Cells

The sensitivity of the assay is limited by the number of cells thatgenerate the nucleic acid template that is used in the amplificationreaction. Typically ˜6 pg of DNA is present in each cell. Therefore tohave a sensitivity of 1/1000,000, ˜6 μg of DNA need to be used. Howeveris peripheral blood only a fraction of the cells are B cells and hence˜6 μg of DNA from peripheral blood may have only 100,000 B cells. Toobtain higher sensitivity higher amounts of DNA can be used. One problemcan be that as more DNA is used the effect of inhibitors purified withthe DNA can be more profound and sample to sample variation may be seen.Obtaining purer population of cells may ameliorate this problem.Generating Peripheral Blood Mononuclear Cell (PBMC) is frequently donein clinical settings. ˜6 μg of DNA from PBMC can have ˜250,000-300,000 Bcells. Capturing B cells specifically can be also done to obtain more Bcells per μg of DNA used.

More than one immune receptor rearrangements can be followed to maximizesensitivity and ameliorate the problem of clonal evolution. Therefore if3 rearrangements are being followed then splitting the available cellsamong them would decrease the sensitivity in the analysis of eachrearrangement. Therefore amplification of the DNA (or RNA) in a waywhere all 3 loci of the rearrangements are amplified before thesplitting ameliorates this problem. Whole genome amplificationmethodologies have been employed previously and can be used here toaccomplish the amplification of the three loci prior to the splitting.Alternatively, amplification for the specific loci in one reaction canbe employed to achieve the same task. In this case a later split toamplify each rearrangement separately is optional. The whole genomeamplification prior to amplification of specific immune receptorrearrangement can also be useful when employed to assess only one immunereceptor rearrangement. For example the assessment of IgH is oftencomplicated with somatic hypermutations making the use of multipleprimer sets often desirable. In this case whole genome amplificationbefore the splitting of the input nucleic acid between differentreactions with different primer sets does not necessarily lead toimproved sensitivity to detect the cancer-specific clonotype. In thiscase the different (e.g 3) reactions assess the full repertoire ofavailable input nucleic acid and hence there would be no advantage ofthe whole genome amplification (aside from preserving the DNA for otherinterrogation). However this is not true when somatic hypermutation atthe sequence complementary to a primer occurs. For example if there isonly one DNA molecule representing the cancer-specific clonotype then itwould go to one of the three reactions. A somatic hypermutation preventsthis specific clonotype to be amplified. On the other hand whole genomeamplification would guard against that since the initial single moleculeof cancer-specific sequence is amplified and would therefore be presentin all 3 tubes. So even though the frequency of the cancer-specificclonotype does not increase in the input template for the locusamplification, the fact that it is present in all 3 tubes is anadvantage. Instead of whole genome amplification locus specificamplification using approaches like long range PCR or using primers fromall three primer sets or preliminary amplification can be done.

Screening for lymphoid neoplasms. The above methods apply to themonitoring of patients after initial diagnosis; however, this inventionapplies also to cancer screening. Screening for primary cancer has beena major force in reducing mortality. Early detection of lymphoidneoplasms may lead to great improvement in the survival rates of thesecancers. It has been shown at least in acute and chronic lymphocyticleukemia that the specific cancer clonotypes can be detected yearsbefore the ultimate diagnosis. It is possible that lymphoma clonotypescan also be detected earlier than is possible using current diagnosticmethodologies. Detection of the cancer clonotypes for screening purposes(i.e., before a primary tumor occurs) can be done using this inventionby sequencing immune cell receptors as described above. The cancerclonotype in each patient is likely to be unique, and it is clear thatone does not know a priori the sequence to be screened for in eachpatient. Many of the methods listed above can, however, be used in bloodfrom a patient who has not yet been diagnosed with cancer in order toidentify clonotypes that are likely to be associated with cancer andtheir levels and changes in these levels can be used to assess the riskof a patient developing clinical cancer.

Types of lymphoid neoplasms. The methods of the provided invention canbe used to monitor lymphoid neoplasms, e.g., lymphoma or leukemia.Mature B cell neoplasms can include, e.g., chronic lymphocyticleukemia/Small lymphocytic lymphoma, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia),splenic marginal zone lymphoma, plasma cell neoplasms (plasma cellmyeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases,and heavy chain diseases), extranodal marginal zone B cell lymphoma(MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicularlymphoma, Mantle cell lymphoma, diffuse large B cell lymphoma,mediastinal (thymic) large B cell lymphoma, intravascular large B celllymphoma, primary effusion lymphoma, and Burkitt lymphoma/leukemia.

Mature T cell neoplasms can include, e.g., T cell prolymphocyticleukemia, T cell large granular lymphocytic leukemia, adult T cellleukemia/lymphoma, extranodal T cell lymphoma (nasal type),enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, mycosisfungoides/Sezary syndrome, primary cutaneous CD30-positive T celllymphoproliferative disorders, primary cutaneous anaplastic large celllymphoma, Lymphomatoid papulosis, Angioimmunoblastic T cell lymphoma,Peripheral T cell lymphoma (unspecified), or Anaplastic large celllymphoma.

The methods of the provided invention can be used to monitor acuteleukemia or chronic leukemia. The leukemia can be acute lymphoblasticleukemia (ALL) (e.g., precursor B acute lymphoblastic leukemia,precursor T acute lymphoblastic leukemia, Burkitt's leukemia, and acutebiphenotypic leukemia); chronic lymphocytic leukemia (CLL) (e.g., B-cellprolymphocytic leukemia); acute myelogenous leukemia (AML) (e.g., acutepromyelocytic leukemia, acute myeloblastic leukemia, and acutemegakaryoblastic leukemia); chronic myelogenous leukemia (CML) (e.g.,chronic monocyte leukemia); hairy cell leukemia; T-cell prolymphocyticleukemia (T-PLL); or large granular lymphocytic leukemia.

I. Cancer Screening

Another example of a situation in which antigen specific T and/or B cellprofiling may be used to inform clinical situations is the use ofspecific cancer autoantigens in cancer screening. Cancer cells oftenproduce aberrant molecules that are antigenic and provoke an immuneresponse. Such molecules can be screened for directly in blood or bodilyfluids in order to assess the likelihood of a patient going on todevelop cancer. The sensitivity of these methods has proven to be alimiting factor however in that these antigens are found in very smallconcentrations in the blood or other bodily fluids when the cancers areat an early stage when they are most likely to be treatable andasymptomatic. These antigenic molecules can, however, provoke an immuneresponse which, though it may be insufficient to control the tumorgrowth, may be of sufficient strength to be measurable as a way ofdetecting early cancer. Antibodies against antigens specific to, forexample, lung and breast cancer cells have been detected and can be usedas a means to screen for these cancers using the antigens themselves tocapture and detect the antibodies (M. Nesterova et al. Biochimica etBiophysica Acta 2006: 1762: 398-403). As described above, these assayslack the ability to differentiate amongst different antibody clones andmiss any potential T cell immune response to the tumor antigens. Themethods described above can be used to enrich for both T cells and/or Bcells that express TCR or BCR that bind antigens known to exist incancer cells. Using the inventions described herein one can generateclonotype frequencies before and after antigen specific T/BCR enrichmentin order to identify T or B cells that are likely to be part of animmune response to these antigens in a particular individual.

There are several ways this invention could be implemented clinically.In the first embodiment, blood from an individual whose risk ofdeveloping a dangerous tumor is to be assessed is sampled and profiledfor full T and B cell clonotype profiles before and after enrichment forcells that bind to a specific set of cancer antigens. These antigenscould be a single antigen species, a set of antigens, or a complexmixture of antigens or could be the entire mix of material from a singletumor of a mix of tumors. These antigens could include p53, c-myc,NY-ESO-1, BRCA1, BRCA2, HER2, MUC1, CAGE, Sox2, GBU4-5, Annexinl, cox-2.Clones that are likely to be associated with an immune response to theseantigens could be identified by a significant shift in frequency beforeand after enrichment, in addition to other parameters including nucleicacid and amino acid sequence parameters, length, segment usage, etc. Therisk of developing cancer could be determined based on a single timepoint measurement of this type or from at least a second time point inwhich changes in the frequency of these clonotypes are used to calculatea risk of cancer. The enrichment could be done at a first time point toidentify the correlating antigen specific clonotypes and thesecorrelating antigen specific clonotypes quantitated in subsequent timepoints in which enrichment is not carried out in order to calculate arisk score.

A population study could also be used to generate an algorithm topredict correlating clonotypes from the antigen specific enrichment. Inthis population study more than one individual with known cancer risksare obtained and antigen specific clonotypes are identified using thetechniques of this invention. This population is used to generate analgorithm which can be used to predict antigen specific clonotypes thatare relevant to the disease in a new individual whose cancer risk isunknown.

J. Adverse Drug Reactions

The benefits of drug treatment are often balanced by their adverseeffects. The majority of adverse drug reactions (ADR) is fairlypredictable and dose dependent. Other ADRs are idiosyncratic, and manyof these are caused by immunological mechanisms. Predisposition toseveral of these ADRs is associated to specific HLA genotypes. Ideallypatients would know ahead of getting the medication that they arepredisposed to have an ADR. Recently the FDA added on the label of theHIV medication, Abacavir, a recommendation to test for allele HLA-B*5701before initiating treatment as patients with this genotype arepredisposed to the drug hypersensitivity reaction. In cases where it isnot possible to tell a patient they are predisposed to have an ADR, itis desirable to detect evidence the ADR before any symptom appear usingblood tests. There are several methods to diagnose immune-related ADR.There are several in vivo methods (Skin testing, intradermal, patchtesting, and drug provocation tests) that reproduce the drug allergy byexposing the patient to the drug. The in vitro methods include assessingbasophil activation, drug-specific IgE, and drug-specific lymphocytestimulation test. Different versions of the drug-specific lymphocyteactivation tests are used to assess different properties of lymphocyteactivation. These include lymphocyte stimulation, lymphocyte migration,lymphocyte toxicity, and lymphocyte transformation tests. Some variantson the tests include the assessment of activation markers like CD69 orthe level of cytokine released. Generally all these methods are used todiagnose patients who already had an allergy and not to predict thehypersensitivity reaction. Additional problems plague the differenttechniques. For example, some of the in vivo tests have some risk ofserious allergic response in the patient. The basophil activation lacksthe specificity to the relevant antigen and drug-specific IgE arerelevant only to those allergy types involving IgE (e.g., hemolyticanemia and anaphylaxis). Therefore there is need for a method that iscapable of predicting ADR either before drug administration or beforesymptoms appear. Assessing the T and/or B cell repertoire can generatesuch a test. Some of the in vitro methods can be used to identify theclonotypes that interact with the drug. For example the lymphocytestimulation test can be done with the specific drug to identifyclonotypes that interact with the drug.

K. Tissue Damage Detection

The use of molecular markers in blood and bodily fluids and/or tissueshas been shown to be able to provide critical information aboutpotentially damaged organs which can point to disease diagnosis andtherapeutic interventions. One example of such a marker is the detectionof the protein troponin in blood as part of the diagnosis of heartdisease. Troponin is a molecule that is highly specific to heart tissueswhich is largely contained within the cells in heart tissue and is foundat very low levels in circulation for individuals with healthy hearts.When heart disease occurs, however, cell death and apoptosis result inthe spilling of this and other molecules into the bloodstream wheresensitive detection using ELISA assays can reveal elevated levels oftroponin that are clearly associated with heart disease.

This paradigm can be readily extended to other tissues that might suffersimilar damage but such techniques are limited by the ability ofresearchers to identify markers that are both sufficiently specific to agiven tissue to provide diagnostically relevant information andsufficiently abundant in early phases of tissue damage to provideinformation at a clinically useful time in the course of a disease thatmight result in organ damage.

It has been shown that while surface markers present on human cells donot result in immune reactions, internally contained molecules withincells can be immunogenic when they are released into the bloodstream. Asin the case of the detection of cancer autoantigens, such organ damagerelated autoantigens might be more sensitively detected indirectlythrough detection of immune cells that are reactive to these antigensthan would be possible by direct antigen detection of the type that isdone in the case of troponin.

This invention thus can be used to provide a diagnostic insight into thelevels of organ damage in an individual. In this embodiment, T and/or Bcells that are reactive to antigens that are specific to molecules foundwithin a particular type of human tissue are enriched using the methodsdescribed above. Shifts in frequency in T and/or B cells clonotypesbefore and after enrichment can be used to ascertain which clonotypesare likely to be reacting to these antigens. This method can be combinedwith sequence algorithms that use sequence parameters to ascertain whichof these enriched clonotypes are most likely to be reacting to theseantigens. Population studies involving more than one individuals withknown organ damage whose antigen specific correlating clonotypes havebeen empirically identified can also be used to inform an algorithm thatcan be used to refine these predictions by identifying sequencecharacteristics that are often associated with these correlatingclonotypes in subsequent individuals.

Antigen specific clonotype correlation can be done at least at a firsttime point to identify and predict correlating clonotypes. Blood orbodily fluids sampled at subsequent time points can then be profiledwith or without antigen specific enrichment to measure the levels ofthese correlating clonotypes which can be used to generate an organdamage score that correlates the degree of damage to a specific organ inthat individual at that time. Levels of these clonotypes can be used toestablish this score as can the shifts in these levels over time.

Antigens for specific tissues can be used in this embodiment. Tissuescould be: heart, lung, liver, intestine, pancreas, esophagus, stomach,kidney, nerves, testes, ovary, prostate, thymus, placenta, uterus, etc.Antigens for each of these tissues could be a select set of geneproducts that are known to be specifically expressed in these tissues.These specifically expressed gene products could be the result oflooking at differential gene expression between these organs and otherorgans. The antigens could be a single antigen, a set of antigens, orcomplex mixtures of materials up to and including material from wholecells from the tissues in question.

L. Identification of Exposure to Local Antigens

In one embodiment of the invention the described methods are used togenerate a database of immune profiles related to a particulargeographic location based upon local antigens. These antigens could bebut are not limited to local pollens. These antigens could have aseasonal component. Once the immune profiles are generated geographiclocations a subject will have his or her current immune profile comparedto the database. Such a comparison is used to determine whether asubject has recently been exposed to the local antigens. In oneembodiment this is method is used to test whether a subject was in asuspected location. In another embodiment this method is used toidentify locations where the subject is likely to have been without apreconceived suspicion of where the suspect has been. Accordingly, amethod of the invention for determining exposure of an individual to oneor more antigens may comprise the steps of (a) determining a profile ofclonotypes from a sample of B cells and/or T cells of the individual,such sample comprising a repertoire of clonotypes thereof; and (b)comparing clonotypes of the profile with clonotypes of anantigen-specific clonotype database to determine a level of clonotypematches, thereby determining a level of exposure to the antigen, theantigen-specific clonotype database including substantially allclonotypes of human TCR and/or immunoglobulin chains specific for theone or more antigens. In one embodiment, the one or more antigensconsist of antigens of a pathogen. In another embodiment, the pathogenis a virus. In another embodiment, such virus is an influenza virus, asmallpox virus, a hepatitis C virus, a coronavirus, a dengue virus, or alentivirus. In one embodiment, such antigen-specific clonotype databaseconsists of clonotypes consisting of substantially all CDR3 regions ofhuman TCRβ and IgH chains.

M. Identification of Exposure to Bio-Terrorism Related Antigens

In one embodiment of the invention the described methods are used togenerate a database of immune profiles related to antigens likely to berelated to the production of bioterrorism related compounds. Theseantigens could be but are not limited to viral vectors capable of beingweaponized. Once the immune profiles are generated subject will have hisor her current immune profile compared to the database. Such acomparison is used to determine whether a subject has recently beenexposed to the bioterrorism related compounds. In one embodiment this ismethod is used to test whether a subject was exposed to a particularsuspected compound. In another embodiment this method is used toidentify whether the subject is likely to have been exposed to a list ofpotential compounds without a preconceived suspicion of which compoundsare expected. In one embodiment the immune profile to be tested isgenerated after a biologic agent has been found. For example, anindividual suspected of a bio-terrorism attack is identified by theauthorities. A sample is obtained from the suspect and an immune profileis obtained using methods described above. This profile is statisticallycompared to a database which contains many sample profiles. The sampleprofiles include immune profiles that represent certain bioterrorismrelated antigens. The sample profiles include immune profiles thatrepresent certain antigens or combinations of antigens that exist onlyin particular geographic locations at particular times of the year. Thecomparison of the suspect's immune profile to this database providesevidence that the suspect was in a particular geographic location duringa particular time frame and that the suspect was exposed to certainbioterrorism related antigens. This evidence is used to further directthe investigation and during prosecution.

Kits

In the commercialization of the methods described herein, kits foramplification of specific somatically rearranged regions or portionsthereof are particularly useful. Such kits may be for carrying out oneor two staged PCRs (as described above) for amplifying a predeterminedsomatically rearranged region or portion thereof for the purpose ofpreparing a sample of clonotypes for sequence analysis. A kit typicallycomprises one or more reagents, such as, without limitation, nucleicacid primers, packaged in a container, such as, without limitation, avial, tube or bottle, in a package suitable for commercial distribution,such as, without limitation, a box, a sealed pouch, a blister pack or acarton.

The package typically contains a label or packaging insert indicatingthat the packaged reagents can be used in a method for generating aclonotype profile from a tissue sample of a patient. As used herein,“packaging materials” includes any article used in the packaging fordistribution of reagents in a kit, including without limitationcontainers, vials, tubes, bottles, pouches, blister packaging, labels,tags, instruction sheets and package inserts. One example of such a kitincludes reagents necessary for the amplification in one tube of TCRβsequences from DNA or RNA extracted T cells of a patient or peripheralblood lymphocytes of a patient or bone marrow of a patient, as describedabove. Another example of such a kit includes reagents necessary for theamplification in a plurality of tubes IgH sequences from DNA or RNAextracted B cells of a patient or peripheral blood lymphocytes of apatient or bone marrow of a patient, as described above. In the latterexample, necessary reagents include a plurality of sets of primers forgenerating nested sets of templates, as described above. Typically, suchplurality is 2 or 3 or 4. For the latter example, in one embodiment,three sets of primers are provided; and more specifically, the followingthree sets of primers are provided: set 1 comprising forward primersfrom Table 5 and reverse primers from Table 8; set 2 comprising forwardprimers from Table 6 and reverse primers from Table 8; set 3 comprisingforward primers from Table 7 and reverse primers from Table 8. Inanother example, the kit would include the above-described reagents,including one or more PCR primer sets and a thermostable DNA polymerase,such as Taq polymerase, and if sequences are amplified from RNA, areverse transcriptase. The primers may be present in quantities thatwould yield a balanced amplification of individual clonotype sequencesin a patient sample, as described above. In one aspect of the invention,quantities of primers are provided to ensure a balanced amplification ofclonotypes. Such balancing of multiplex PCRs is well known bypractitioners of ordinary skill in the art and includes, but is notlimited to, adjusting the concentrations of primers in the reactionand/or selecting the positions and lengths of primers in a region ofinterest to increase or decrease the rate of annealing of individualprimers. In one embodiment, the quantities of primers are selected sothat in the PCR their concentrations are such that the rate at whicheach primer anneals to its primer binding site is substantiallyidentical. In another embodiment, quantities of primers are selected sothat each sequence in a sample is amplified to an amount that is within2-fold of the average amplification amount of a random sample ofclonotypes. In still another embodiment, such random sample contains atleast 100 clonotypes.

Thermostable DNA polymerases and transcriptases are commerciallyavailable from a variety of manufacturers. Additional materials in thekit may include: suitable reaction tubes or vials, a barriercomposition, typically a wax bead, optionally including magnesium;reaction mixtures (often concentrated, for example 2×, 5×, 10× or 20×)for the PCR stages, including necessary buffers and reagents such asdNTPs; nuclease- or RNase-free water; RNase inhibitor; control nucleicacid(s) (i.e. such as internal standards), and/or any additionalbuffers, compounds, co-factors, ionic constituents, proteins andenzymes, polymers, and the like that may be used in multiplex PCRreactions.

Components of a kit are packaged in any manner that is commerciallypracticable. For example, PCR primers and/or reverse transcriptase maybe packaged individually to facilitate flexibility in configuring theassay, or together to increase ease of use and to reduce contamination.Similarly, buffers, salts and co-factors can be packaged separately ortogether. The kits also may include reagents and mechanical componentssuitable for the manual or automated extraction of nucleic acid from atissue sample. These reagents are known to those skilled in the art andtypically are a matter of design choice. For instance, in one embodimentof an automated process, tissue is disrupted ultrasonically in asuitable lysis solution provided in the kit.

Example 1 Determining the State of an Autoimmune Disease

A sample of cerebral spinal fluid (CSF) and blood is taken from apatient with an episode peak of multiple sclerosis. CD4+ cells areisolated from the CSF and blood, and the CDR3 of the T cell receptor βgene is amplified by PCR. The amplified fragments are further amplifiedto add bridge amplification primer binding sites and sequencing primerbinding sites for Solexa sequencing. The variable regions of the T cellreceptor β gene are sequenced to identify the clonotypes. The sequenceinformation is used to generate a clonotype profile for the patient.

Another blood sample is taken when the patient is at a relativelyinactive state of multiple sclerosis. The same procedure as above isrepeated to generate a clonotype profile. Pathological clonotypes areidentified as those that are high at the peak episode and went downsignificantly at the inactive state. Another blood sample is taken fromthe patient at a later state. At this time only a fraction of the T cellreceptor β gene CDR3 regions are amplified and then sequenced. Thissubset contains the pathological clonotypes. The level of the variousclonotypes is determined to assess the disease state of the patient.

Example 2 TCRβ Repertoire Analysis Amplification and Sequencing Strategy

In this example, TCRβ chains are analyzed. The analysis includesamplification, sequencing, and analyzing the TCRβ sequences. One primerAGCGACCTCGGGTGGGAACA (SEQ ID NO: 1) is complementary to a commonsequence in Cβ1 and Cβ2, and there are 34 V primers (Table 1) capable ofamplifying all 48 V segments. Cβ1 or Cβ2 differ from each other atposition 10 and 14 from the J/C junction. The primer for Cβ1 and Cβ2ends at position 16 bp and has no preference for Cβ1 or Cβ2.

The 34 V primers are modified from an original set of primers disclosedin Van Dongen et al, U.S. patent publication 2006/0234234, which isincorporated herein by reference.

TABLE 1  Primer sequences complementary to the different V families.V segment family Primer Sequence SEQ ID NO V20-1 AACTATGTTTTGGTATCGTCAGT2 V29-1 TTCTGGTACCGTCAGCAAC 3 V9, 5-1, 5-6, 5-5, 5-8, 5-4AAGTGTATCCTGGTACCAACAG 4 V9, 5-1, 5-6, 5-5, 5-8, 5-4BAGTGTGTACTGGTACCAACAG 5 V9, 5-1, 5-6, 5-5, 5-8, 5-4CACTGTGTCCTGGTACCAACAG 6 V9, 5-1, 5-6, 5-5, 5-8, 5-4DAGTGTGTCCTGGTACCAACAG 7 V9, 5-1, 5-6, 5-5, 5-8, 5-4ETCTGTGTACTGGTACCAACAG 8 V7-3, 7-6, 7-9, 7-2, 7-4A CCCTTTACTGGTACCGACAG 9V7-3, 7-6, 7-9, 7-2, 7-4B GCCTTTACTGGTACCGACAG 10V7-3, 7-6, 7-9, 7-2, 7-4C CCCTTTACTGGTACCGACAAA 11 V7-8, 16ATTTTGGTACCAACAGGTCC 12 V7-8, 16B TTTTGGTACCAACAGGCCC 13 V 7-7AACCCTTTATTGGTATCAACAG 14 V4-1, 4-3, 4-2A CGCTATGTATTGGTACAAGCA 15V4-1, 4-3, 4-2B CGCTATGTATTGGTACAAGCA 16 V12-3, 12-4, 12-5TTTCTGGTACAGACAGACCATGA 17 V3-1 TACTATGTATTGGTATAAACAGGACTC 18 V25-1CAAAATGTACTGGTATCAACAA 19 V28, 10-3, 6-2, 6-3, 6-1, 6-6, 24-1ACAAAATGTACTGGTATCAACAA 20 V28, 10-3, 6-2, 6-3, 6-1, 6-6, 24-1BATGTACTGGTATCGACAAGACC 21 V6-4, 6-9A TGCCATGTACTGGTATAGACAAG 22V6-4, 6-9B ATACTTGTCCTGGTATCGACAAG 23 V10-1, 10-2,6-5, 6-9,6-8, 27AATATGTTCTGGTATCGACAAGA 24 V10-1, 10-2,6-5, 6-9,6-8, 27BATATGTCCTGGTATCGACAAGA 25 V10-1, 10-2,6-5, 6-9,6-8, 27CACATGTCCTGGTATCGACAAGA 26 V14 TAATCTTTATTGGTATCGACGTGT 27 V19GCCATGTACTGGTACCGACA 28 V18 TCATGTTTACTGGTATCGGCAG 29 V30CAACCTATACTGGTACCGACA 30 V11-1, 11-3,11-2A CATGCTACCCTTTACTGGTACC 31V11-1, 11-3,11-2B CACAATACCCTTTACTGGTACC 32 V2ATACTTCTATTGGTACAGACAAATCT 33 V13 CACTGTCTACTGGTACCAGCA 34 V15CGTCATGTACTGGTACCAGCA 35

The Illumina Genome Analyzer is used to sequence the amplicon producedby the above primers. A two-stage amplification is performed onmessenger RNA transcripts (200), as illustrated in FIGS. 2A-2B, thefirst stage employing the above primers and a second stage to add commonprimers for bridge amplification and sequencing. As shown in FIG. 2A, aprimary PCR is performed using on one side a 20 by primer (202) whose 3′end is 16 bases from the J/C junction 204) and which is perfectlycomplementary to Cβ1 203) and the two alleles of Cβ2. In the V region(206) of RNA transcripts (200), primer set (212) is provided whichcontains primer sequences complementary to the different V regionsequences (34 in one embodiment). Primers of set (212) also contain anon-complementary tail (214) that produces amplicon (216) having primerbinding site (218) specific for P7 primers (220). After a conventionalmultiplex PCR, amplicon (216) is formed that contains the highly diverseportion of the J(D)V region (206, 208, and 210) of the mRNA transcriptsand common primer binding sites (203 and 218) for a secondaryamplification to add a sample tag (221) and primers (220 and 222) forcluster formation by bridge PCR. In the secondary PCR, on the same sideof the template, a primer (222 in FIG. 2B and referred to herein as“C10-17-P5”) is used that has at its 3′ end the sequence of the 10 basesclosest to the J/C junction, followed by 17 bp with the sequence ofpositions 15-31 from the J/C junction, followed by the P5 sequence(224), which plays a role in cluster formation by bridge PCR in Solexasequencing. (When the C10-17-P5 primer (222) anneals to the templategenerated from the first PCR, a 4 bp loop (position 11-14) is created inthe template, as the primer hybridizes to the sequence of the 10 basesclosest to the J/C junction and bases at positions 15-31 from the J/Cjunction. The looping of positions 11-14 eliminates differentialamplification of templates carrying Cβ1 or Cβ2. Sequencing is then donewith a primer complementary to the sequence of the 10 bases closest tothe J/C junction and bases at positions 15-31 from the J/C junction thisprimer is called C′). C10-17-P5 primer can be HPLC purified in order toensure that all the amplified material has intact ends that can beefficiently utilized in the cluster formation.)

In FIG. 2A, the length of the overhang on the V primers (212) ispreferably 14 bp. The primary PCR is helped with a shorter overhang(214). Alternatively, for the sake of the secondary PCR, the overhang inthe V primer is used in the primary PCR as long as possible because thesecondary PCR is priming from this sequence. A minimum size of overhang(214) that supports an efficient secondary PCR was investigated. Twoseries of V primers (for two different V segments) with overhang sizesfrom 10 to 30 with 2 bp steps were made. Using the appropriate syntheticsequences, the first PCR was performed with each of the primers in theseries and gel electrophoresis was performed to show that all amplified.In order to measure the efficiency of the second PCR amplification SYBRgreen real time PCR was performed using as a template the PCR productsfrom the different first PCR reactions and as primers Read2-tag1-P7 andRead2-tag2-P7. A consistent picture emerged using all 4 series of realtime data (2 primary PCRs with two different V segments and twosecondary PCR with different primers containing two different tags).There was an improvement in efficiency between overhang sizes 10 and 14bp. However there was little or no improvement in efficiency with anoverhang over 14 bp. The efficiency remained high as the overhang becameas small as 14 bp because of the high concentration of primers allowingthe 14 bp to be sufficient priming template at a temperature much higherthan their melting temperature. At the same time the specificity wasmaintained because the template was not all the cDNA but rather a lowcomplexity PCR product where all the molecules had the 14 bp overhang.

As illustrated in FIG. 2A, the primary PCR uses 34 different V primers(212) that anneal to V region 206) of RNA templates 200) and contain acommon 14 by overhang on the 5′ tail. The 14 bp is the partial sequenceof one of the Illumina sequencing primers (termed the Read 2 primer).The secondary amplification primer (220) on the same side includes P7sequence, a tag (221), and Read 2 primer sequence (223) (this primer iscalled Read2_tagX_P7). The P7 sequence is used for cluster formation.Read 2 primer and its complement are used for sequencing the V segmentand the tag respectively. A set of 96 of these primers with tagsnumbered 1 through 96 are created (see below). These primers are HPLCpurified in order to ensure that all the amplified material has intactends that can be efficiently utilized in the cluster formation.

As mentioned above, the second stage primer, C-10-17-P5 (222, FIG. 2B)has interrupted homology to the template generated in the first stagePCR. The efficiency of amplification using this primer has beenvalidated. An alternative primer to C-10-17-P5, termed CsegP5, hasperfect homology to the first stage C primer and a 5′ tail carrying P5.The efficiency of using C-10-17-P5 and CsegP5 in amplifying first stagePCR templates was compared by performing real time PCR. In severalreplicates, it was found that PCR using the C-10-17-P5 primer had littleor no difference in efficiency compared with PCR using the CsegP5primer.

Amplicon (300) resulting from the 2-stage amplification illustrated inFIGS. 2A-2B has the structure typically used with the Illumina sequenceras shown in FIG. 3A. Two primers that anneal to the outmost part of themolecule, Illumina primers P5 (AATGATACGGCGACCACCGAG) (SEQ ID NO: 36)and P7 (CAAGCAGAAGACGGCATACGAGAT) (SEQ ID NO: 37) are used for solidphase amplification of the molecule (cluster formation). Three sequencereads are done per molecule. The first read of 100 bp is done with theC′ primer, which has a melting temperature that is appropriate for theIllumina sequencing process. The second read is 6 bp long only and issolely for the purpose of identifying the sample tag. It is generatedusing the Illumina Tag primer (AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC) (SEQID NO: 38). The final read is the Read 2 primer, an Illumina primer withthe sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 39). Usingthis primer, a 100 by read in the V segment is generated starting withthe 1st PCR V primer sequence.

A set of 6 bp sequence tags to distinguish different samples run in thesame sequencing lane was designed, where each tag is different from allthe other tags in the set by at least 2 differences. The 2 differencesprevent misassignment of a read to the wrong sample if there is asequencing error. The alignment done to compare the tags allowed gapsand hence one deletion or insertion error by sequencing will also notassign the read to the wrong sample. Additional features in selectingthe tags was to limit single base runs (4 A or T and 3 G or C) as wellas no similarity to the Illumina primers. In total 143 tags weregenerated, 96 of them are used.

Sequencing TCRβ. Six multiplexed amplifications with the pooled oligosand one cDNA sample as a template were used. Three of each of theamplifications were done with Accuprime and another three with highfidelity Taq. Two amplifications with each enzyme used cDNA thatcorrespond to 500 ng initial RNA, and one amplification with each enzymeused 10 times less cDNA. For each of the six reactions a primary andsecondary PCR was performed and the amplified material was sequencedusing the Illumina platform and the scheme described above. 100 bpsequence from each side was obtained. The primary analysis of the datawas done using the same concepts described below.

To assess reproducibility of the assay it was determined whetherclonotype levels are consistent in the duplicate experiments. As shownin FIGS. 5A-5C, high correlation is obtained when the same enzyme andstarting input cDNA amount was used (each of the 2 comparisons hadr2=0.944). When different enzymes were used the correlation gets worse(median correlation for the 4 possible combinations r2=0.931), and it isonly modestly reduced (r2=0.924) when the 2 enzymes were used to amplifysmaller input cDNA (corresponding to only 50 ng RNA).

In FIGS. 5A-5C, identical sequences in each sample were identified. Thento deal with sequencing errors some clonotypes were coalesced to formlarger clonotypes using the general approaches described in the sectionof primary analysis of sequence. The counts of clonotypes were thencomputed in each sample. A fraction of the clonotypes (not shown in thefigure) were present in one sample but not another, likely due to thealgorithm coalescing them with another clonotype in one sample but notthe other. The frequency of clonotypes in a sample is then computed asits number of counts divided by the total number of reads obtained forthat sample. For example if 1,000 counts are observed for a clonotype ina sample with 1,000,000 reads, its frequency is computed as 0.1%. FIG.7A shows the log₁₀ of the frequency of each clonotype in the twoduplicate samples using Accuprime and cDNA corresponding to 500 ng ofRNA as input template. The correlation (r²) between these duplicates is0.944. FIG. 7B depicts the log₁₀ of the frequency of each clonotypeusing cDNA corresponding to 500 ng of RNA as input template andAccuprime (X axis) or High fidelity Taq (Y axis). There are 4comparisons with this combination with a median correlation r²=0.931.The one shown in the figure has r²=0.929. FIG. 7C shows the log_(o) ofthe frequency of each clonotype using cDNA corresponding to 50 ng of RNAas input template and Accuprime (X axis) or High fidelity Taq (Y axis).The observed correlation r2=0.924.

Example 3 IgH Repertoire Analysis Amplification and Sequencing Strategy

In this example, three primers are used to amplify V regions of IgHmolecules. Preferably, the primers are in regions avoiding the CDRs,which have the highest frequency of somatic mutations. Three differentamplification reactions are performed. In each reaction, each of the Vsegments is amplified by one of the three primers and all will use thesame C segment primers. The primers in each of the separate reactionsare approximately the same distance from the V-D joint and differentdistances with respect to the primers in different reactions, so thatthe primers of the three reactions are spaced apart along the V segment.Assuming the last position of the V segment as 0, then the first set ofprimers frame A) have the 3′ end at approximately −255, the second setframe B) have the 3′ end at approximately −160, and the third set frameC) have the 3′ end at approximately −30. Given the homology betweenseveral V segments, to amplify all the 48V segments and the many knownalleles (as defined by the international ImMunoGeneTics informationsystem <<http://imgt.cines.fr/>>) 23, 33, and 32 primers in the A, B,and C frames respectively, is needed. The list of primers are shown inTables 2, 3, and 4.

TABLE 2  Frame A Primers Segment Sequence SEQ ID NO IGHV1_1CCTCAGTGAAGGTCTCCTGCAAGG 40 IGHV1_2 CCTCGGTGAAGGTCTCCTGCAAGG 41 IGHV1_3CCTCAGTGAAGGTTTCCTGCAAGG 42 IGHV1_4 GGGCTACAGTGAAAATCTCCTGCAAGG 43IGHV2_1 AAACCCACACAGACCCTCACGCTGAC 44 IGHV2_2 AAACCCACAGAGACCCTCACGCTGAC45 IGHV2_3 AAACCCACACAGACCCTCACACTGAC 46 IGHV3_1CTGGGGGGTCCCTGAGACTCTCCTG 47 IGHV3_2 CTGGGGGGTCCCTTAGACTCTCCTG 48IGHV3_3 CAGGGCGGTCCCTGAGACTCTCCTG 49 IGHV3_4 CAGGGCCGTCCCTGAGACTCTCCTG50 IGHV3_5 CTGGGGGGTCCCTGAAACTCTCCTG 51 IGHV3_6CTGGCAGGTCCCTGAGACTCTCCTG 52 IGHV3_7 CTGGAGGGTCCCTGAGACTCTCCTG 53IGHV3_8 CTGGGAGGTCCCTGAGACTCTCCTG 54 IGHV3_9 TGGGGGGGCCCTGAGACTCTCCT 55IGHV4_1 CTTCGGAGACCCTGTCCCTCACCTG 56 IGHV4_2 CTTCGGACACCCTGTCCCTCACCTG57 IGHV4_3 CTTCACAGACCCTGTCCCTCACCTG 58 IGHV4_4CTTCGGAGACCCCGTCCCTCACCTG 59 IGHV4_5 CGGGGACCCTGTCCCTCACCTG 60 IGHV5_1GATCTCCTGTAAGGGTTCTGGATACAGCT 61 IGHV6 TCGCAGACCCTCTCACTCACCTGTG 62

TABLE 3  Frame B Primers Segment Sequence SEQ ID NO IGHV6TGGATCAGGCAGTCCCCATCGAGAG 63 IGHV5_1 GCTGGGTGCGCCAGATGCCC 64 IGHV2_1TGGATCCGTCAGCCCCCAGG 65 IGHV2_2 TGGATCCGTCAGCCCCCGGG 66 IGHV1_1GTGCGACAGGCCCCTGGACAA 67 IGHV1_2 GGGTGCGACAGGCCACTGGACAA 68 IGHV1_3GTGCGCCAGGCCCCCGGACAA 69 IGHV1_4 GGGTGCGACAGGCTCGTGGACAA 70 IGHV1_5GGGTGCAACAGGCCCCTGGAAAA 71 IGHV1_6 GGGTGCGACAGGCTCCTGGAAAA 72 IGHV1_7GTGCGACAGGCCCCCGGACAA 73 IGHV1_8 GTGCGACAGGCCCCCAGACAA 74 IGHV4_1TCCGCCAGCCCCCAGGGAAGG 75 IGHV4_2 TCCGGCAGCCCCCAGGGAAGG 76 IGHV4_3TCCGGCAGCCACCAGGGAAGG 77 IGHV4_4 TCCGCCAGCACCCAGGGAAGG 78 IGHV4_5TCCGGCAGCCCGCCGGGAA 79 IGHV4_6 TCCGGCAGCCGCCGGGGAA 80 IGHV4_7TCCGGCAGCCCGCTGGGAAGG 81 IGHV4_8 TCCGCCAGCCCCTAGGGAAGG 82 IGHV3_1GGTCCGCCAGGCTCCAGGGAA 83 IGHV3_2 GTTCCGCCAGGCTCCAGGGAA 84 IGHV3_3GGTCCGCCAGGCTTCCGGGAA 85 IGHV3_4 GGTCCGTCAAGCTCCGGGGAA 86 IGHV3_5GATCCGCCAGGCTCCAGGGAA 87 IGHV3_6 GGTCCGCCAAGCTCCAGGGAA 88 IGHV3_7GGTCCGCCAGGCTCCAGGCAA 89 IGHV3_8 GGTCCGCCAGGCTCCAGGCAA 90 IGHV3_9GGTCCGCCAGGCTCCGGGCAA 91 IGHV3_10 GGGTCCGTCAAGCTCCAGGGAAGG 92 IGHV3_11CTGGGTCCGCCAAGCTACAGGAAA 93 IGHV3_12 GGTCCGCCAGCCTCCAGGGAA 94 IGHV3_13GGTCCGGCAAGCTCCAGGGAA 95

TABLE 4  Frame C Primers Segment Sequence SEQ ID NO IGHV7CTAAAGGCTGAGGACACTGCCGTGT  96 IGHV6 CTCTGTGACTCCCGAGGACACGGCT  97IGHV5_1 AGTGGAGCAGCCTGAAGGCCTC  98 IGHV2_1 TGACCAACATGGACCCTGTGGACAC  99IGHV1_1 ACATGGAGCTGAGCAGCCTGAGATC 100 IGHV1_2 ACATGGAGCTGAGCAGGCTGAGATC101 IGHV1_3 ACATGGAGCTGAGGAGCCTGAGATC 102 IGHV1_4ACATGGAGCTGAGGAGCCTAAGATCTGA 103 IGHV4_1 GAGCTCTGTGACCGCCGCGGAC 104IGHV4_2 GAGCTCTGTGACCGCCGTGGACA 105 IGHV4_3 GAGCTCTGTGACCGCTGCAGACACG106 IGHV4_4 GAGCTCTGTGACCGCTGCGGACA 107 IGHV4_5GAGCTCTGTGACTGCCGCAGACACG 108 IGHV4_6 GAGCTCTGTGACTGCAGCAGACACG 109IGHV4_7 GAGCTCTGTGACTGCCGCGGACA 110 IGHV4_8 GAGCTCTGTGACCGCGGACGCG 111IGHV4_9 GGCTCTGTGACCGCCGCGGAC 112 IGHV4_10 GAGCTCTGTGACCGCCGCAGACA 113IGHV4_11 GAGCTCTGTGACCGCTGACACGG 114 IGHV3_1CAAATGAACAGCCTGAGAGCCGAGGACA 115 IGHV3_2 CAAATGAACAGCCTGAAAACCGAGGACA116 IGHV3_3 CAAATGAACAGTCTGAAAACCGAGGACA 117 IGHV3_4CAAATGATCAGCCTGAAAACCGAGGACA 118 IGHV3_5 CAAATGAACAGTCTGAGAACTGAGGACACC119 IGHV3_6 CAAATGAACAGTCTGAGAGCCGAGGACA 120 IGHV3_7CAAATGAACAGCCTGAGAGCTGAGGACA 121 IGHV3_8 CAAATGAGCAGCCTGAGAGCTGAGGACA122 IGHV3_9 CAAATGAACAGCCTGAGAGACGAGGACA 123 IGHV3_10CAAATGGGCAGCCTGAGAGCTGAGGACA 124 IGHV3_11 CAAATGAACAGCCTGAGAGCCGGGGA 125IGHV3_12 CAAATGAACAGTCTGAGAGCTGAGGACA 126 IGHV3_13CAAATGAGCAGTCTGAGAGCTGAGGACA 127On the C segment side, two sequences with one base difference betweenthem (GCCAGGGGGAAGACCGATGG (SEQ ID NO: 128), and GCCAGGGGGAAGACGGATGG)(SEQ ID NO: 129) cover the four segments and the multiple known allelesof IgG. A scheme similar to the two stages of PCR for TCRβ genes isused.

On the V side, the same 5′ 14 bp overhang on each of the V primers isused. In the secondary PCR, the same Read2-tagX-P7 primer on the V sideis employed. On the C side a strategy similar to that used with TCRβamplification is used to avoid variants among the different IgG segmentsand their known alleles. The primer sequence(AATGATACGGCGACCACCGAGATCTGGGAAGACGATGGGCCCTTGGTGGA) (SEQ ID NO: 130)comprises the sequence of the C segment from positions 3-19 and 21-28and it skips position 20 that has a different base in at least one ofthe different IgG alleles and the sequence for P5 that is can be usedfor formation of the clusters as shown in FIG. 4A.

A multiplexed PCR using three pools of primers corresponding to thethree frames was carried out using cDNA as a template. After primary andsecondary PCRs, the products were run on an agarose gel. Single bandswith the appropriate relative sizes were obtained from the three pools.

In one embodiment, three different reactions from a single sample aremixed at equimolar ratio and subjected to sequencing. Sequencing is donefrom both directions using the two Illumina primers, such as describedabove. 100 bp is sequenced from each side. The maximal germ linesequences encompassing the D+J segments are ˜30 bp longer for BCR thanTCR. Therefore if the net result of nucleotide removal and addition atthe joints (N and P nucleotides) generate a similar distribution for IgHand TCRβ, on average 90 bp and maximally 120 bp of sequence after the Csegment is sufficient to reach the 3′ of the V segment. Therefore, inmost cases, the sequence from the C primer is sufficient to reach the Vsegment. Sequencing from one of the Illumina adapters identifies the Vsegment used as well as somatic hypermutations in the V segments.Different pieces of the V segments are sequenced depending on which ofthe three amplification reactions the sequence originated from. The fullsequence of the BCR can be aligned from different reads that originatedfrom different amplification reactions. The sequencing reaction from theone end showing the full CDR3 sequence greatly facilitates the accuratealignment of different reads.

Example 4 TCR and IgH Repertoire Analysis in SLE Patient Samples

It will first be tested whether there are clonotypes that correlate withdisease activity in patients. Second, a set of sequence characteristicsand/or cell surface markers that distinguish clonotypes that correlatewith disease from those that do not is defined. Third, the degree towhich clonotype analysis provides clinically useful information ismeasured, such as the correlation with short term (e.g., 3 month)outcome.

1. Presence of Clonotypes Correlating with Disease

There is two main tasks: identifying correlating clonotypes andmeasuring disease activity from their level. These tasks can be done ina clinical setting in two steps for each patient:

1) A Calibration test can be done to determine the identity of thecorrelating clonotypes for the specific patient. This can be done bysequencing IgH and TCRβ RNA or linked TCRα-TCRβ sequence from a singlecell) for each patient at a time of a peak of an episode, at which timethe correlating clonotype level can reach their highest levels.

2) A Monitoring test can be done to determine the level of thecorrelating clonotypes at a time point subsequent to the calibrationtest. This can be done by sequencing IgH and TCRβ RNA and determiningthe level of the specific correlating clonotypes that had beenidentified in the calibration sample of the same patient. The level ofthe correlating clonotypes is used to compute the disease activity atthese points.

Amplification, sequencing, and primary analysis development as describedabove is used to assess patient samples. Specifically, a set of systemiclupus erythematosus (SLE) patients is assessed that have a one yearfollow up period and serial blood samples during this period. Thesepatients were seen By Dr. Michele Petri at Johns Hopkins Medical Schoolevery three months for one year, and clinical measures of diseaseactivity including Systemic Lupus Erythematosus Disease Activity Index(SLEDAI), Physician Global Assessment (PGA), as well as multiple labtests including C3 (Complement 3) and anti-ds DNA levels are availablefor all visits of all patients. Drugs being administered to thepatients, include prednisone, plaquenil, NSAID, NSAIDType,acetylsalicylic acid (ASA) dose, plavix, diuretic, ACE-Inhibitors orangiotensin receptor blockers (ARBs), Ca channel blocker, Triam and,solumedrol, Patients who had at least at one time during the follow up asignificant change in disease activity as defined by a 3 points changeon the SLEDAI or a 1 point change in PGA is studied. Overall there are181 patients (with a total of 815 blood samples) who fit these criteria.RNA from all these blood samples is subjected to multiplex PCR usingprimers described above to amplify the sequences that encompass CDR3 inIgH and TCRβ. All the amplified materials is sequenced to a millionreads) and the abundance of different clonotypes is determined

Using the clinical data, sequencing, characteristics that distinguishclonotypes whose level correlate with disease activity from those thatdo not is identified. Second, an algorithm to determine disease activityusing the blood IgH and TCRβ profile is developed.

2. Identification of Characteristics of Correlating Clonotypes

It is anticipated that clonotypes that are relevant to the disease isincreased at the time of high disease activity. However, not allenriched clonotypes at a point of high disease activity necessarilycorrelate with disease. For example, in a particular patient there mightbe 10 enriched clonotypes at the point of high disease activity, butonly 5 correlate with the disease. In order to identify these relevantclonotypes, a subset of clonotypes that are clearly correlating withdisease and another set that clearly do not correlate with disease isstudied. Characteristics that distinguish those two classes ofclonotypes is investigated.

All patients will have at least one significant change in diseaseactivity during the one year follow up in this experimental design. TheIgH and TCR clonotypes obtained at the peak of disease activity in eachpatient is analyzed. Sets of correlating and not correlating clonotypesamong those with the highest level clonotypes is selected. Hence thefirst step is to define clonotypes that are at a high level. Thespecific criteria to choose the clonotypes that will enter the analysiswill include a combination of frequency rank of the clonotype and thelevel of clonotype (number of clonotype reads per million), as well asevidence the clonotype does not belong to the distribution of lowfrequency clonotypes.

This set of clonotypes from each patient sample, termed High PrevalentClonotypes (HPC) is further analyzed. The correlation of the level ofeach of these clonotypes with clinical measures is evaluated. Thecorrelation of SLEDAI score with the clonotype level is computed. Foreach patient there is 4-5 study points that can be used to assess thecorrelation of SLEDAI with the level of each HPC. The distribution ofthese obtained correlations is investigated. It is anticipated that mostof the HPCs will have low correlation with SLEDAI. It is investigatedwhether at the high correlation end there is an excess to what isexpected to be generated randomly. For example with 4 and 5 data pointsit is expected that ˜2.5% and ˜0.6% of the correlation levels (r²)is >0.9 by chance. A higher proportions of HPCs with r²>0.9 indicatesthe presence of a clonotypes that correlate with disease. In addition tocomparing the number of correlating clonotypes with random expectation,a permutation analysis is performed where the correlation of SLEDAIscores from one patient and the level of individual HPCs from another iscalculated. The distribution of correlations generated from thispermutation can be used as the “background” correlation. To ensure itsvalidity, it is confirmed that there is little correlation betweenSLEDAI between different patients). Excess correlation at the highcorrelation end, e.g., r²>0.9 will indicate the presence of clonotypesthat correlate with disease. The highest correlating clonotypes as theset of correlating clonotypes is picked. Because the number of HPCs thathas a by chance correlation higher than a set threshold is known (fromcalculation using random assumption or through the permutation analysisdescribed above), the threshold to define the correlating clonotype canbe set in such a way as to have 10% false discovery rate, i.e. 10% ofthe correlating clonotypes set is correlating by chance. A set of HPCsthat have very little correlation with SLEDAI score is picked. Thosewill serve as the set of non-correlating clonotypes. These 2 sets ofclonotypes can be further analyzed to identify characteristics that maydistinguish them. These characteristics can then be looked for in newsamples to identify the clonotypes likely to be correlating with diseaseactivity in these samples. The blood levels of these clonotypes can thenbe followed to determine disease activity.

One complication arises from the premise that clonotype level may changebefore disease activity does. Hence it is possible that by attempting tostudy only HPCs that highly correlate with SLEDAI, clinically usefulclonotypes that change earlier than SLEDAI may be eliminated. Anotherset of clonotypes is picked that correlate with a Modified SLEDAI(MSLEDAI) score. MSLEDAI is the same as SLEDAI in all the study pointsexcept those just before a significant change. For those data points theMSLEDAI score is the average between the SLEDAI score at that point andthe next study point. Clonotypes that change before SLEDAI are likely toshow better correlation to MSLEDAI than SLEDAI. It is informative tocompute the excess number of HPCs that have high correlation withMSLEDAI than expected by random or permutation generated expectations.

Characteristics that distinguish correlating clonotypes from those thatdo not correlate will then be identified. The analysis is done in theexact manner for those clonotypes that correlate with SLEDAI or MSLEDAI.In either case the goal would be for these set of characteristics tocorrectly recapitulate this classification enabling the identificationof correlating clonotypes in the next set of samples. It is expectedthat each patient will have a unique set of correlating clonotypes, butthe training study is designed to generate the rules that predict thecorrelating clonotypes from a calibration sample (at high diseaseactivity). Two general types of parameters can be tested: those that areobtained from the sequencing data itself, and those that can use extraexperimentation. Extra experimentation can include the assessment ofdifferent cells with different cell surface or other markers. Here are afew types of parameters that is investigated: 1) Sequence motif: Themotif can be a specific V or J region, a combination VJ, or shortsequences in DJ region that is associated with a clonotype beingcorrelating. 2) Size of the clonotype. 3) Level: Absolute level (numberof reads per million) or rank level. 4) Similarity to other clonotypes:The presence of other highly related clonotypes, like those with silentchanges (nucleotide differences that code for same amino acids) or thosewith conservative amino acid changes. 5) For the BCRs the level ofsomatic mutations in the clonotype and/or the number of distinctclonotypes that differ by somatic mutations from some germ lineclonotypes.

Each of these parameters is individually studied for association withcorrelating clonotypes. A threshold of 0.05 (uncorrected for multipletesting) is set to eliminate factors that are not likely to contributeto prediction of correlating clonotypes. Given the multiple parameters,many tests is performed to generate multiple positive results by chance.However the main goal of this step is to filter the parameters to asmaller set. The set of positive parameters will then be used to createan algorithm to classify the two sets of clonotypes. A machine learningalgorithm is employed that uses the different parameters to classify thetwo sets of clonotypes. In order to minimize the risk of overfitting,the cross validation technique is used. Using this algorithm eachclonotype will get a score that corresponds to the likelihood it is acorrelating clonotype. A threshold will then be placed to classifyclonotypes above it as correlating and those below it asnon-correlating. The accuracy of the classification can be estimated bythe cross validation technique; for example, the clonotypes are put inequal groups and the algorithm using all clonotypes except one group.Clonotypes in the last group (test group) are then classified using thealgorithm that was obtained using the rest of the clonotypes. This isiterated as many times as the number of groups, and in each iterationall the groups except one are used for training and one group isclassified. The accuracy of the algorithm can be estimated from theaverage accuracy of the different classifications in the differentiterations. It is of note that in all these iterations the exactalgorithm would be slightly different. The accuracy of classification isthen an estimate as it is not on the final algorithm but rather on a setof related algorithms generated with training data from all clonotypesexcept one.

Ultimately, two algorithms is generated trained on two differentcorrelating clonotypes sets: one correlating with SLEDAI and the othercorrelating with MSLEDAI. Even if the clonotypes in the training set aredifferent the resulting algorithm may or may not be very different,depending on whether these clonotypes indeed come from two distinctpopulations. The algorithms is compared. Additionally these algorithmsis used to identify correlating clonotypes that were not initially inthe training set. The clonotypes identified in the two algorithms iscompared, and if the initial clonotypes in the two training sets werefrom the same population, the identified clonotypes are likely to bevery similar Unless the results of the algorithm were quite similar,both algorithms is carried to identify correlating clonotypes in orderto measure lupus disease activity.

Other experimental approaches can add to the power of sequencing inidentifying clonotypes that correlate with diseases. Correlatingclonotypes may be enriched in cells with some surface or other markers.For example B cells with high levels of CD27 are known in active lupuspatients, and hence it might be that correlating clonotypes might beenriched in the CD27 population of cells. If that is borne out to betrue, prediction of correlating clonotypes can be improved by doing anenrichment for cells with high levels of CD27. Specifically, asequencing reaction can be performed on the IgH sequences from all Bcells in the blood sample as well as from those B cells with high CD27.Correlating clonotypes are expected to be present at higher frequency inthe high CD27 population than in the all blood sample.

3. Using IgH and TCRβ Profiles to Determine Lupus Disease Activity

The section above described clonotype-based analysis to identifyfeatures of correlating clonotypes. In addition, for that analysis onlya fraction of all the HPCs were used to clearly designate clonotypes ascorrelating or non-correlating. This section describes analysis that isat the patient level aiming to compute a measure of disease activity, tobe called AutoImm (AI) score. The algorithm developed per the abovesection is applied to identify correlating clonotypes among all theHPCs. The level of these correlating HPCs is determined. The level ofthe correlating clonotypes can be normalized to the total number of TCRclonotypes as well as to HPCs predicted not to correlate with disease.The level of these correlating clonotypes at different time points isused to compute AI score at these different points.

In patients with more than one correlating clonotypes, the informationregarding the level of these different clonotypes is combined. Inaddition data from IgH and TCRβ clonotypes is integrated. Differentalgorithms for making the combination is attempted. For example, theaverage, median, sum, and highest correlating clonotype level isstudied. The clonotype level can be its simple linear read counts, thelogarithm of that or some other conversion. It can potentially be thedifference between correlating and non-correlating clonotypes.Furthermore methods for weighted average can be utilized. The weightingcan be based on the likelihood of a clonotype to be correlating.

In order to evaluate which of the models is optimal, all the models isassessed to identify the one that generates the highest correlationbetween the AI score and the SLEDAI score. For this analysis thecorrelation of SLEDAI and AI scores is done across all the data obtainedfrom all the study points from all patients. In order to estimate andameliorate the degree of overfitting, the cross validation technique isused. The level of correlation measured reflects the “cross sectional”relationship between the AI and SLEDAI scores. In addition to SLEDAI,the correlation with other clinical measures like C3 and anti-ds DNAantibody levels as well as urine protein/serum creatinine for patientswith kidney manifestation and blood counts for patients withhematological involvement is studied. The correlation may be due to theclassification of patients into high and low disease activity, and isnot necessarily a reflection of AI correlating with SLEDAI score withina patient. To demonstrate that, “longitudinal” assessment is done.

4. Longitudinal Analysis

In the longitudinal analysis, two general questions is assessed: does AIscore at one study point predict disease activity at the same point, anddoes AI score at one study point predict disease activity at a laterpoint, e.g., the next study point 3 months later.

The relationship between AI and SLEDAI scores at the same study point isassessed in two ways. First the correlation of the AI and SLEDAI in eachpatient is calculated, and then the average and median patientcorrelation level is computed. If the correlation seen in crosssectional analysis above is due to classification of high and lowdisease activity patients and not changing disease activity withinindividual patients, then the longitudinal correlation in individualpatients is likely to be low. A high median patient correlation levelsuggests that AI does reflect the SLEDAI score at an individual patientlevel. In addition to the correlation of AI and SLEDAI scores, thecorrelation of AI with other relevant measures like C3 and anti-ds DNAantibody is assessed as well as urine protein/serum creatinine forpatients with kidney manifestation and blood counts for patients withhematological involvement.

Another way to demonstrate the ability of AI score to measure diseaseactivity changes in individual patients is by determining its accuracyin distinguishing states of high from low disease activity in the samepatients. For each of the 181 patients, the two study points when theSLEDAI where at the highest (to be called HDAP for high disease activitypoint) and lowest levels (to be called LDAP for low disease activitypoint) is selected. The distribution of the AI of all the HDAPs withthat of the AI of all the LDAPs is compared, and the p-value that theyare different is computed. In addition, the frequency that the AI atHDAP is higher than LDAP in each patient is assessed. If AI does notchange with disease activity in an individual patient then it isexpected that AI at HDAP is higher than that at LDAP only 50% of times.Another analysis is done where the fraction of times that AI at HDAP ishigher than that at LDAP by a meaningful difference (i.e., above thelikely AI variation) is determined. To measure the fluctuation of AI,all the study points from all the patients is used, and the standarddeviation (and relative standard deviation) of AI in the different binsof SLEDAI values can be computed. This will generate relative standarddeviation across all patients (AI-RSDall) and this value may or may notbe dependent on SLEDAI (i.e. the AI-RSDall may be different at differentSLEDAI values). The proportion of patients where AI at HDAP is higherthan AI at LDAP by a specific number (e.g., 2) of AI-RSDall can becomputed. There can be some systematic bias where the computed AI insome patients is consistently higher (or lower) than what is expectedfrom the SLEDA score. Therefore AI-RSDall is a combination of theintrinsic fluctuation of AI within a patient as well as the systematicdifference of AI for patients with similar SLEDAI. The intrinsicfluctuation of AI can be computed within a patient by calculating thestandard deviation (and relative standard deviation) of AI scores amongstudy points with similar SLEDAI values (<2 points difference) within apatient. The median among all the patients of the relative standarddeviation can be computed (AI-RSDpt-med). The proportion of patientswhere AI at HDAP is higher than AI at LDAP by a specific number (e.g.,2) of AI-RSDpt-med can then be evaluated.

After demonstration that AI does indeed fluctuate with SLEDAI withinindividual patients it is evaluated whether AI can predict SLEDAI at thenext study point, 3 months later. To assess that correlation levelbetween the AI score at time 0 and the SLEDAI score at time+3 months canbe quantitated. The correlation can be computed on a patient level andthen the median patient correlation can be obtained. Another way todemonstrate the ability of AI to predict near future disease activity isto evaluate the sensitivity and specificity of AI in predicting diseaseactivity 3 months in the future. Clinically, those patients who aredoing well on their current management can be distinguished from thosethat do not. A patient state at a particular time is classified into oneof two classes: Poor Control (PC) and include patients who in 3 monthswill have high disease activity (SLEDAI >6 points) and/or a flare(SLEDAI increase by 3 points), and Good Control (GC) and includepatients who in 3 months will have low or moderate with disease activity(SLEDAI <6) and/or a significant reduction in disease activity (SLEDAIdecrease by 3 points). The classification sensitivity can then beevaluated and specificity obtained using different thresholds of AI. AROC curve that describes the performance of AI in predicting the stateof the patient (PC or GC) can be generated 3 months ahead of time. Theperformance obtained by this test is compared with that of standardclinical measures including SLEDAI, anti-ds DNA and C3 levels.

An analysis to evaluate the ability of AI to predict changes in SLEDAIscores 3 months later will also conducted. Using data from all studypoints of all patients, the relationship between AI and SLEDAI scorescan be plotted to identify the “cross sectional” correlation level asdiscussed above. This determines the relationship between SLEDAI and AIat the same study point. This relationship is fit with an equationallowing the prediction of the SLEDAI score given an AI score (or viceversa). If AI predicts flares then changes in SLEDAI at some study point1 is preceded by changes in AI at point 0. Therefore, if a flare occursbetween point 0 and 1, the AI score at point 0 (to be called AImeas) ishigher than what is expected (to be called Alexp) given the SLEDAI atstudy point 0. On the other hand with no change in disease activitybetween the study point 0 and study point 1, the AI score at point 0 isvery similar to what is expected given the SLEDAI at study point 0. Therelative AI change (Rel-AI-diff) can be computed by dividing thedifference of AImeas and AIexp by AImeas. The sensitivity andspecificity of AI in predicting a significant change in SLEDAI 3 monthslater can be evaluated by using different thresholds of Rel-AI-diff. Thethresholds can be bidirectional so if the Rel-AI-diff at a specificstudy point is higher than a specific threshold a flare is predicted,and similarly if it is lower than the negative of the specific thresholda significant reduction in SLEDAI is expected. On the other hand whenthe Rel-AI-diff at a study point is between the threshold and itsnegative, no significant changes in disease activity is expected. A ROCcurve showing the trade of sensitivity and false positives can begenerated using many different thresholds of Rel-AI-diff. Similar ROCcurves can be generated using standard clinical measures includingSLEDAI, anti-ds DNA and C3 levels.

If the fluctuation of AI varies at different SLEDAI values, the aboveanalysis is refined. A section above described the computation ofAI-RSDall and AI-RSDpt-med and mentioned evaluating whether they changeat different SLEDAI values. If they do then the ROC analysis can be doneas described above but instead of using different thresholds ofRel-AI-diff, different thresholds of AI-RSDall and AI-RSDpt-med is used.The performance obtained by the test with that of standard clinicalmeasures including SLEDAI, anti-ds DNA and C3 levels is compared.

In the above analysis, attempts are made to predict the SLEDAI at point1 from the AI score at point 0. It is likely that in addition to theabsolute level at point 0, the change of AI from point −1 to 0 isinformative in predicting SLEDAI at point 1. For example consider apatient who has at study point −1 an AI score of X-1, and at point 0 theAI score is increased to a new value X0 that is appreciably higher thanX-1. This patient may have higher likelihood of a flare at point 1 thana patient whose AI has been stable at X0 at study points −1 and 0. Thisconcept of AI change or velocity is incorporated to generate a ModifiedAI (MAI) score. To generate a MAI at point 0 the AI score at point −1and at point 0 is needed, and hence one data point per patient will nothave an MAI associated with it. The specific formula to incorporate thevelocity into AI calculation to obtain MAI is optimized. Thisoptimization may be done through maximization of the correlation of MAIand SLEDAI three months later. The cross validation design is used toevaluate and control the degree of overfitting. Correlation can be donefor data points of all samples, but also can be done at a patient leveland the median correlation among all patients can be assessed. Thelatter approach ameliorates the issue of some patients having asystematic bias of too low or too high AI score. Using MAI, the sametype of ROC analysis that was mentioned for AI can be performed toassess its ability to predict SLEDAI 3 months later. First, analogouslyto what is described for AI, an analysis can be done to show the abilityof MAI at point 0 to distinguish PC and GC states at point 1.Additionally, an analysis similar to what was described for AI to assessthe ability of MAI at point 0 can be performed to predict significantdisease activity change (3 points change on SLEDAI) between points 0and 1. For this latter analysis different thresholds of Rel-AI-diff,AI-RSDall or AI-RSDpt-med can be used. The performance of MAI iscompared with that of AI to determine whether the addition of thevelocity factor is useful.

One complication of the described study is that treatment changes aredone for different patient during the follow up period of the study.This is likely to complicate the prediction of disease activity. Forexample, consider two patients with the same AI score at point 0 and oneof those patients had a reduction in medication at the same time. Thelikelihood of this patient to have a rise in disease activity at point 1is then likely to be higher than for the patient who did not changemedications at point 0. This is likely to lead to underestimation of theperformance of AI. One way to alleviate that is to eliminate all thepoints with significant medication changes from the study. Another is tomodify the AI score to include whether a patient has a medication changeand create a medication-modified AI. So in the example above with thetwo patients, the one with the medication change will have a highermedication-modified AI.

5. Integration with Other Predictive Markers

The predictive ability of the disease activity marker can be maximized.Therefore the predictive ability of the TCR/BCR repertoire informationintegrated with other markers is tested. These markers include standardmarkers used in the clinic like anti-ds DNA and C3 levels. It will alsoinclude other markers that are published. For example a panel ofchemokines has already been shown to have some predictive ability usingthe same set of patients as is used. Whether this panel will increasethe predictive ability of the TCR and BCR repertoire is evaluated. Thefirst step is to integrate the AI score with the additional measure togenerate an Expanded AI (EAI) score. Different ways to do theintegration can be assessed, and this can be optimized throughmaximization of the correlation of EAI and SLEDAI three months later.The cross validation design is used to evaluate and control the degreeof overfitting. Using EAI the ability to predict disease activity 3months later is assessed by its ability to distinguish GC from PC and topredict changes in disease activity. The performance in measuringdisease activity and change in disease activity can be described throughROC analysis as described above.

6. Validation

The number of variables being tested is high compared with the number ofsamples. This can lend itself to overfitting, with initially promisingresults not being able to be validated in later studies. A crossvalidation approach is used in the training to get a measure of theextent of overfitting. However, a validation on an independent set ofsamples is involved in later work. This is not part of this proposal,but this marker can be clinically applicable. Using the data obtainedabove, it can be determined whether AI, MAI, or EAI, should be validatedand the specific way to compute the measure of interest. One specificalgorithm is taken for validation. In addition one or more specificendpoints is specified. The sensitivity and specificity of AI can beassessed in the ability to distinguish GC from PC 3 months later toevaluate the ability of AI to predict disease activity. In anotherexample the sensitivity and specificity of AI to predict significantdisease activity change in 3 month using a specific Rel-AI-diffthreshold can be assessed.

Example 5 Measuring Response of an SLE Patient to Drug Therapy

The methods of the provided invention is used to measure the response ofan SLE patient to drug therapy. Determination of whether an SLE patientbeing given an expensive drug with serious side effects is responding tothe drug plays a role in both patient care and also for making theadministration of such care cost effective. Many clinical indicators ofdisease activity respond to treatment imprecisely and after a time lagof up to several months. During this time, disease may progress and sideeffects may add complications to therapy. A prompt understanding of thedrug response would allow patients to be switched to more effectivetherapies more rapidly.

In this Example, a 35 year old African American female with a priordiagnosis of lupus presents to her regular rheumatologist. The patient'sdisease status is assessed on a quarterly basis through a comprehensiveclinical assessment in addition to laboratory testing includingmeasurement of 0, anti-ds DNA antibody levels, blood counts, andurinalysis. During one visit the patient complains of skin lesions andfatigue, and urinalysis shows evidence of proteinuria and/or cell casts.The rheumatologist refers the patient to a nephrologist for a kidneybiopsy to assess inflammatory status of the kidney and orders serumcreatinine and 24 hour urine protein to creatinine ratio to assess thedegree of the impairment of the kidney function. A kidney biopsy showsevidence of diffuse lupus nephritis, while the urine protein tocreatinine test reveals evidence of nephrotic syndrome (urine protein tocreatinine ratio of 3.6). Based on this information a diagnosis of acutelupus nephritis is given and the patient is begun on a course of drugtherapy. There are several possible drugs that can be chosen at thispoint. Immunomodulators such as mycophenolate mofetil (Cellcept) areoften used although sometimes in severe cases drugs such asMethotrexate, Azathiopurine (Imuran) Cyclophosphamide (cytoxan), areprescribed. Rituximab (Rituxan) is also sometime used as a second orthird choice. One of these drugs is often used in combination with asystemic steroid such as Prednisone or methylprednisolone in order tosuppress the acute symptoms. Here, mycophenolate mofetil is prescribedat 150 mg per day alongside 60 mg of prednisone. Given the many sideeffects of steroids, including the risk of osteoporosis, hyperglycemia,weight gain, and other Cushingoid symptoms in the long term, thepatient's prednisone dose is tapered over ˜6 weeks if the clinicalpicture allows that.

The first question that is determined is whether the patient isresponding to therapy, and as a result, can the dose of steroid can beappropriately decreased. Therefore, during this period the patient'sserum creatinine as well as urine protein and creatinine are followed toensure the patient is responding to the medications. Frequent kidneybiopsy can be done to detect whether the inflammatory damage is beingreversed; however, routine use of kidney biopsy carries too great a riskand is too invasive to be practical. Current blood based markers thatare being used to assess inflammatory status are of limited use inmaking this decision in that they are not sufficiently well correlatedwith underlying disease to be relied upon to risk the increased sideeffects that accompany high doses of steroids. Serum and urine functionmarkers may have some delay in detecting improvement in inflammatorystatus and hence steroids may be tapered before these markers show adefinitive change and hence extending the period of the renal flare. Aslower taper, informed by more sensitive markers, in these cases couldhave shortened the flare period preventing further damage to kidneytissue. After the reduction of steroid to a maintenance dose ofapproximately 10 mg the patient may show persistently elevated levels ofprotein in the urine and the high urine protein to creatinine ratio of2, and the physician must now decide whether to switch from Cellcept toanother medication. Arguing in favor of this is the continued evidenceof loss of kidney function but without an accurate measure ofinflammatory kidney status, it can be difficult to know whether thedisease itself is in remission having nevertheless done some level ofirreversible kidney damage that is resulting in these persistent levelsof proteinuria. Here again the existing blood based markers areimperfectly informative and a further kidney biopsies are not practical.This decision would be greatly aided by an accurate blood based measureof disease status.

AutoImm Load would be very helpful in this situation to assess theresponse to therapy by measuring disease activity either alone or incombination with other markers of disease activity. An algorithm forAutoImm Load is developed using the study described above. Thecorrelating clonotypes that is used to calculate AutoImm Load ismeasured using a calibration test. This calibration test is done usingblood from a patient at a time of peak disease activity, for example atthe start of therapy. The calibration test is performed using blood oralternatively using the tissue that is affected (e.g. kidney biopsy orskin biopsy). At a later time at which the response to therapy is to beassessed, a blood sample is taken and used along with the calibrationtest to measure AutoImm Load. This is used to make a treatment decision.If the correlating clonotypes are derived from a populations study,there is no need for the calibration test and a blood test at the timeat which the response to therapy is to be assessed is sufficient tomeasure AutoImm Load in order to inform the treatment decision.

Example 6 Determination of Appropriate Time to Taper or Stop Therapy foran SLE Patient

The methods of the provided invention can be used to determine theappropriate time to taper or stop therapy for an SLE patient. Inaddition to the time lag that can be exhibited by the clinical measuresof disease activity, a further difficulty lies in the lack ofsensitivity of these measurements. Subclinical disease can nonethelessresult in a re-flaring of the disease if therapy is tapered too early.As a result of this, courses of immunosuppressant therapy are typicallyadministered for a time period that is much longer than is necessary forthe average patient to ensure that the risk of re-flaring is low for theaverage patient yet may still be long enough for the tail end ofdistribution. Therefore significant over-treatment, causing side effectsand costs are occurring in most patients, while under-treatment of somepatients occurs causing potentially preventable re-flares. A method thatcould measure subclinical activity that was predictive of the risk ofre-flaring would allow therapy to be tapered based on such measuresinstead of relying on overtreatment by design.

In this example, the patient from Example 7 is on prednisone andmycophenolate mofetil for a period of 6 months and urine protein tocreatinine ratio returns to a level of 0.5. This level remains above thebaseline level expected in healthy individuals but it is not clear thatthis level is not due to some kidney damage that is not reversible.Other clinical measures of inflammation are normal and the patient doesnot report any other symptoms. At the same time the patient isexperiencing moderate levels of nausea and weight gain as possible sideeffects to the medications that additionally have serious long term sideeffects. The doctor is faced with a difficult decision: balancing thefear of tapering the Cellcept and/or steroid too quickly, which couldresult in renewed kidney inflammation and likely further long termirreversible kidney damage and the adverse reactions that can occur dueto the medications. Here again an unambiguous assessment of the diseasestatus without having to perform a kidney biopsy would play a role inmaking this decision. Attempt of reducing steroids is recommendedthrough repeated trials of steroids leading to the recurrence of thesame clinical dilemma. In fact this question arises at every time thepatient is in remission and the patient is on steroids orimmunomodulators.

AutoImm Load would be very helpful in this situation to assess whetheror not to taper therapy by measuring disease activity either alone or incombination with other markers of disease activity. An algorithm forAutoImm Load is developed using the study described above. Thecorrelating clonotypes that is used to calculate AutoImm Load ismeasured using a calibration test. This calibration test is done usingblood from a patient at a time of peak disease activity, for example atthe start of therapy. The calibration test could be performed usingblood or alternatively using the tissue that is affected (e.g. kidneybiopsy or skin biopsy). At a later time at which the level of diseaseactivity is to be assessed, a blood sample can be taken and used alongwith the calibration test to measure AutoImm Load. This is used to makea treatment decision and to evaluate whether the patient has anydetectable disease activity. If the correlating clonotypes are derivedfrom a populations study, there is no need for the calibration test anda blood test at the time at which the response to therapy is to beassessed is sufficient to measure AutoImm Load in order to inform thetreatment decision.

Example 7 Prediction of Flares in an SLE Patient

One challenge in treating SLE patients is that represented by the factthat flares arise without warning, thus thwarting the physicians'efforts to treat the disease preventively. Waiting for flares to occurbefore beginning treatment subjects patients to potentially destructiveclinical symptoms, can involve expensive and inconvenienthospitalization, and may cause long term organ damage to be done whilealso necessitating aggressive therapeutic interventions that arethemselves fraught with side effects. A much more desirable paradigmwould be a therapeutic paradigm in which flares are detected at asubclinical phase at which time therapy could be administeredproactively saving significant suffering to the patient, resulting inless expensive hospitalizations and ultimately enabling better long termprognosis for the patients.

The patient from Example 7 is recovering from the acute flare describedabove, and the patient is tapered off of all therapies except Plaquiniland a low dose of 5 mg of Prednisone. Nevertheless this patient remainsat a high risk of having another inflammatory episode. As a result, thispatient will remain in the care of a rheumatologist who will continuefollowing patient's clinical symptoms and laboratory tests.Unfortunately these symptoms and tests do not provide early warning foran imminent flare until patients actually have exhibited clinicalsymptoms of a flare and the sequence repeats itself. A highly specificmarker of increasing subclinical activity could be included in theroutine clinical assessment of the patient in order to detectunambiguous signs of a flare which may reach a clinically detectablestage within the subsequent 1-3 months. Beginning therapies earliermight make the flare less severe and may allow treatment to beaccomplished with less long term organ damage or less steroids used thanwhat is currently the case.

AutoImm Load would be very helpful in this situation to assess thelikelihood of an incipient flare by measuring disease activity eitheralone or in combination with other markers of disease activity. Thisscore either by itself or the rate of increase (velocity) oracceleration of this score can be used to assess the likelihood ofprogression to a flare. An algorithm for AutoImm Load could be developedusing the study described above. The correlating clonotypes that is usedto calculate AutoImm Load could be measured using a calibration test.This calibration test could be done using blood from a patient at a timeof peak disease activity, for example at the start of therapy. Thecalibration test could be performed using blood or alternatively usingthe tissue that is affected (e.g. kidney biopsy or skin biopsy). At alater time at which the response to therapy is to be assessed, a bloodsample can be taken and used along with the calibration test to measureAutoImm Load. This can be used to make a treatment decision. If thecorrelating clonotypes are derived from a populations study, there is noneed for the calibration test and a blood test at the time at which theflare risk is to be assessed is sufficient to measure AutoImm Load inorder to inform the treatment decision.

Example 8 Objective Measure to Assess Subjective Symptoms of SLEPatients

SLE affects many organs and produces many potential symptoms includingones that are very common in the healthy populations. For example, if anSLE patient complains of a headache, the headache may be a sign of CNSlupus or can be due to the common headache. Similarly, if SLE patientscomplain of worsening fatigue over a period of time, the worseningfatigue may be due to deterioration of their disease or can be due todepression or other causes. The availability of an objective measurethat reflects disease activity can be of great help in the management ofSLE patients.

The patient in Example 7 presents to the rheumatologist with chiefcomplaints of headache, fatigue, and difficulty with concentration.Patient's headache is recurrent and only transiently gets better withMotrin treatment. The patient's SLE is otherwise in good control.Relevant psychosocial stressors in the patient's life include that sheis going through divorce. Physicians are in a dilemma when they face SLEpatients with symptoms that are non-specific to SLE and are common inthe general population. Is the patient suffering from CNS lupus? Orcould she suffering from other common causes of her symptoms, likedepression? Current laboratory tests currently lack the sensitivity andspecificity to be relied on to distinguish these possibilities. Areliable test to measure SLE disease activity can be utilized routinelyto help in distinguishing the two possibilities.

AutoImm Load would be very helpful in this situation to objectivelyassess the disease activity either alone or in combination with othermarkers of disease activity. An algorithm for AutoImm Load is developedusing the study described above. The correlating clonotypes that is usedto calculate AutoImm Load will be measured using a calibration test.This calibration test is done using blood from a patient at a time ofpeak disease activity, for example at the start of therapy. Thecalibration test is performed using blood or alternatively using thetissue that is affected (e.g. kidney biopsy or skin biopsy). At a latertime at which the objective disease activity is to be assessed, a bloodsample can be taken and used along with the calibration test to measureAutoImm Load. This is used to make a treatment decision. If thecorrelating clonotypes are derived from a populations study, there is noneed for the calibration test and a blood test at the time at which theobjective disease activity is to be assessed is sufficient to measureAutoImm Load in order to inform the treatment decision.

Example 9 Measuring Response to Drug Therapy of an MS Patient

As stated above, one of the principle challenges in MS therapy ismeasuring how well and whether a patient is responding to a drugtherapy. During progressive and late stage disease there are clinicalassessments such as the Expanded Disability Status Score (EDSS) whichmeasure the degree of physical impairment that has resulted from thedisease. However, these assessments are not useful in early stage orrelapsing/remitting disease. Clinical parameters around relapses can beused to assess disease progression, but these are coarse and laggingindicators, as patients can go several years between relapses, duringwhich little evidence can be gleaned from clinical assessments. Lastly,brain imaging such as gadolinium enhanced MRI can be used to examinebrain lesions. MS patients are typically given such an MRI on a yearlybasis. However, such images lack specificity. Furthermore, as a measureon integrated brain damage, they are not good measures of currentdisease activity but rather reflect the history of the disease and itsimpact on the brain.

While it is true that the current clinical treatment paradigm for MS isthat patients diagnosed with relapsing remitting disease should be undercontinuous therapy in order to delay the onset of progressive disease,the increasing repertoire of approved drugs to treat MS makes the lackof biological feedback increasingly problematic. The list shown above ofapproved drugs to treat MS continues to get longer as the substantialinvestment in MS therapies begins to bear fruit. Each of these drugs hasserious side effects and is very expensive to administer, with costsfrom $30,000-$100,000 per year of treatment. Patients that are not wellmanaged will sooner transition to progressive disease which isdebilitating and causes expensive health care interventions includinghospitalizations and long term care. Hence, the patient can be allowedto receive optimal therapy early in treatment.

Clinical Utility Example

Patient profile: A 30 year old female comes to the hospital withmonocular visual impairment with pain. She is given a neurologicalassessment and a lumbar puncture to obtain cerebral spinal fluid whichis used to assess whether clonal T cells are present. She also isreferred for a brain MRI. Based on these tests, a diagnosis of MS ismade. She is prescribed Betaseron 250 mcg per injection to beself-administered subcutaneously every other day. At a follow-up visitsix months later, the patient is complaining of depression and weightgain. No further neurological events have been reported to thephysician. The doctor is now faced with a clinical dilemma. Should thedoctor maintain the therapy as it is been administered? Should a newtherapy be used? Should the doctor order an MRI incurring cost andsubjecting the patient to additional contrast exposure? Should thedoctor wait until the next scheduled MRI shows new lesions? Should thedoctor wait to see if flares recur? All of these decisions would benefitfrom an unambiguous measure of whether the disease is active or not.

AutoImm Load would be very helpful in this situation to assess theresponse to therapy by measuring disease activity either alone or incombination with other markers of disease activity. An algorithm forAutoImm Load is developed using the studies described herein. Thecorrelating clonotypes that is used to calculate AutoImm Load ismeasured using a calibration test. This calibration test is done usingblood from a patient at a time of peak disease activity, for example atthe start of therapy. The calibration test could be performed usingblood or alternatively using the tissue that is affected (e.g. CSF). Ata later time at which the response to therapy is to be assessed, a bloodsample can be taken and used along with the calibration test to measureAutoImm Load. This can be used to make a treatment decision. If thecorrelating clonotypes are derived from a population study, there is noneed for the calibration test and a blood test at the time at which theresponse to therapy is to be assessed is sufficient to measure AutoImmLoad in order to inform the treatment decision.

Example 10 Prediction of MS Flares

As in all autoimmune diseases, the amelioration of flares is a principlegoal of therapy. Not only are flares debilitating for the patient andexpensive to treat, but it is increasingly believed that each flarecontributes to longer term non reversible disease progression. Severaltherapies can be used to control incipient flares such as IVmethylprednisolone or oral prednisone. Such medications have significantside effects and as such are not prescribed without evidence of anactive flare. A measure of increasing subclinical activity that wascorrelated with subsequent clinical flares could be used to inform thissort of proactive flare treatment which could result in shorter and lessdamaging flares. In addition there are therapies that demonstrate highclinical efficacy for reduction of flares that carry risks of verysignificant and lethal of side effects. One such drug is Tysabri, a drugthat has been shown to result both in improved clinical outcomes and toincrease the risk of deadly brain infections such as PML. These riskshave reduced the value of such drugs to last line therapy when otherdrugs are proving to no longer control progression and limited the valueof these drugs as chronic treatments. A test that could predict when theflare state is incipient could increase the utility of such drugs asthey could be used in a manner similar to steroids to control acuteflare periods while minimizing the risks of lethal side effects.

Clinical Utility Example

The patient from Example 11 is on Betaseron for 3 years and reports aclinical flare that lasts a week. The patient's MRI at the end of theyear shows significant new lesions multiple discrete variable sizedovoid perpendicularly directed T2W and FLAIR hyperintense lesions(plaques), appearing iso-hypointense on T1W images and hyperintense onT2W images involving bilateral periventricular and subcortical whitematter regions, including the calloso-septal interface). The doctor isconcerned that the patient is at high risk of flares over the course ofthe next 12 months. A clinical dilemma presents itself. Does the doctorwait for further clinical symptoms to intervene with additional therapy?Should the doctor switch therapies? If so, should another class ofinjectable be used such as copaxone or should a new class of therapy beused such as Tysabri? Should steroids be prescribed? A test that couldmonitor sub clinical disease activity and show when the disease isincreasing and when a flare is likely to result could be used to helpmake these clinical decisions.

AutoImm Load would be very helpful in this situation to assess the riskof flare by measuring disease activity either alone or in combinationwith other markers of disease activity. An algorithm for AutoImm Loadcould be developed using the studies described in this invention. Thecorrelating clonotypes that is used to calculate AutoImm Load could bemeasured using a calibration test. This calibration test could be doneusing blood from a patient at a time of peak disease activity, forexample at the start of therapy. The calibration test could be performedusing blood or alternatively using the tissue that is affected (e.g.CSF). At a later time at which the risk of flare is to be assessed, ablood sample can be taken and used along with the calibration test tomeasure AutoImm Load. This can be used to make a treatment decision. Ifthe correlating clonotypes are derived from a population study, there isno need for the calibration test and a blood test at the time at whichthe flare risk is to be assessed is sufficient to measure AutoImm Loadin order to inform the treatment decision.

Example 11 Monitoring Therapy Compliance for MS

Because of the relative infrequency of clinical symptoms in the earlystages of the disease, the interactions between a patient and his or herphysician are not very frequent. At the same time, the therapies thatare being prescribed are both expensive and inconvenient for thepatient, involving self injections that can cause painful reaction andside effects. There is as a result a significant degree of noncompliancewith therapeutic regimes which are hard for a physician to monitor asthe interactions between the patient and doctor is not routine. A testthat could measure the state of the sub clinical disease would allowboth doctor and patient to see on a routine basis how well controlledthe underlying disease is. Such methods have proved very effective inHIV patients in motivating them to pursue therapy effectively. A testblood test that was performed quarterly would allow the physician to seethe patient and measure the state of the disease.

AutoImm Load would be very helpful in this situation to assess thecompliance with therapy by measuring disease activity either alone or incombination with other markers of disease activity. An algorithm forAutoImm Load is developed using the studies described herein. Thecorrelating clonotypes that is used to calculate AutoImm Load ismeasured using a calibration test. This calibration test is done usingblood from a patient at a time of peak disease activity, for example atthe start of therapy. The calibration test could be performed usingblood or alternatively using the tissue that is affected (e.g. CSF). Ata later time at which the compliance with therapy is to be assessed, ablood sample is taken and used along with the calibration test tomeasure AutoImm Load. This is used to make a treatment decision and tobetter guide the patient toward better compliance. If the correlatingclonotypes are derived from a population study, there is no need for thecalibration test and a blood test at the time at which the compliancewith therapy is to be assessed is sufficient to measure AutoImm Load inorder to inform the treatment decision.

Example 12 Amplification of Mouse TCRβ and IgH Sequences

An amplification and sequencing scheme for mouse TCRβ and IgH isdeveloped that is similar to that developed for humans. Similar methodsto minimize the differences in amplification efficiency of differentsequences and similar validation techniques using spikes and the 5′ RACEtechnique described above is applied. The minimum input amount of cDNAis determined in a similar methodology as described for human samples.One difference in the amplification scheme between mouse and humans isthat the two C segments for TCRβ in mouse do not have any polymorphismsin the 50 bp closest to the J/C junction. Therefore, in the scheme theprimer for the first stage amplification is placed at positions 25-50and for the second stage amplification the primer is placed at positions1-25, and the primer will have a 5′ tail for the latter primercontaining the P5 sequence. The different sequences will improvespecificity and is similar to the strategy used in humans except thereis no need to “loop out” any bases for polymorphisms.

Example 13 Primary Analysis of Mouse Sequence Data

The analysis framework that is used for analysis of mouse data issimilar to that described above for the human data. One difference isthat the mouse samples is sequenced to less depth than the humansamples. It is anticipated that the blood samples from the mouse is 1004In 100 μl of blood there are ˜100K lymphocytes and hence sequencing to adepth much higher than 100K does not significantly improve theprecision. Therefore, only 100K reads for each mouse sample is obtained.Even though the number of reads is smaller for mouse than humans, alarger fraction of mouse total and blood lymphocytes is sampled. Thenumber of total mouse lymphocytes is expected to be more than 3 ordersof magnitude smaller than that of humans. Similarly 100 μl of blood willprovide a better sampling (˜10%) of the lymphocytes in the mouse bloodat the time when compared to sampling obtained using 10 ml of humanblood (0.2%).

Example 14 IgH and TCR Repertoire Analysis in Mouse SLE Model

A mouse model of SLE is used to study the relationship between TCR/BCRrepertoire and disease activity. The mouse model is the B6 with the sle1and sle3 loci from NZM2410. These B6.sle1.sle3 (BSS) mice developSLE-like nephritis in a spontaneous fashion. Three types of cohorts isstudied. For all study points, blood BUN, creatinine, and anti-nuclearautoantibodies, urine protein, and creatinine level is obtained. It isdetermined whether a score generated from blood TCR/BCR repertoirecorrelates well with these measured indices of kidney disease. The firstcohort is similar to the human cohort described where longitudinal bloodsamples is collected along with kidney function assessment.Specifically, 7 BSS mice is followed on a monthly basis till month 8. Atthe end, these mice is sacrificed and in addition to blood, spleen andkidney tissue are analyzed. As a control, 5 B6 mice is assessed in asimilar manner. The second cohorts is cross sectional where differentcohorts of animals is sacrificed at specific times and spleen, kidney,and blood samples is analyzed at that time. Specifically, 5 BSS mice issacrificed each month and blood, spleen, and kidney is analyzed. As acontrol, two B6 control mice is assessed in the same fashion. Finally athird cohort is treated with steroids after disease onset and nephritisassessment and blood samples obtained on a regular basis after that.Specifically at 4 months of age, 20 mice that have the disease istreated with steroids and then on a biweekly basis for the next 4 monthsblood is taken for TCR/BCR repertoire analysis and kidney functionassessment. As a control 5 BSS mice is treated with placebo and followedin a similar fashion. TCR and BCR repertoire analysis is performed fromall the study points (i.e. different time points and different tissuesfor the same time point). The analysis will involve 2 stage PCR,sequencing processing, and primary data analysis as described above.

Example 15 Identification and Dynamics of Clonotypes that Correlate withMouse SLE

First, a set of clonotypes that correlate with renal function isidentified. As a measure of renal function, urine protein/creatinineratio, serum creatinine, or BUN levels can be used. In the first andthird cohorts, the correlation of the blood level of each HPC clonotypewith each of the three measures can be assessed. In a similar manner towhat is described in humans, it can be assessed whether there is a greatincrease in the number of clonotypes with high correlation to 1, 2, orall 3 of the renal function measures over random expectation (orpermutation testing). Given that random expectation, the correlationthreshold is picked where only 10% of the clonotypes with a correlationlevel above that threshold are expected to have the observed correlationlevel by chance (10% false discovery). These clonotypes is focused on,and this set is defined as “correlating clonotypes”.

In addition to this statistical method to identify correlatingclonotypes, clonotypes might be identified relevant to disease by a“functional” method of enrichment of specific clonotypes in kidneytissue. By the functional method a set of clonotypes may be identifiedin cohort 2 that may be relevant to disease, and these is calledfunctionally-identified correlating clonotypes. The extent of overlapbetween the “statistical” definition and the “functional” definition ofcorrelating clonotypes can be assessed. Cohorts 1 and 3 have kidneysamples collected at the last time point. It can be assessed whetherclonotypes enriched in these kidney samples are present in the blood andare among the clonotypes with higher correlation with renal function.

The dynamics of correlating clonotypes (statistically and functionallyidentified) can then be evaluated. For example, using data from cohort2, the time course of the rise and fall (if any) of their levels isevaluated in the three compartments: kidney, blood, and spleen.

In the statistically identified correlating clonotypes, a subset of thecorrelating clonotypes would be identified by virtue of theircorrelation with renal function. The correlating clonotypes can beidentified without knowing the renal function data. In other words, thecharacteristics that distinguish the correlating clonotypes from thosethat are irrelevant to disease can be understood. In order to do that aset of clonotypes with low correlation to renal function is identifiedas control non correlating clonotypes.

Characteristics of clonotypes that correlate with disease. Afteridentification of the two sets of clonotypes, correlating and notcorrelating, characteristics that distinguish these two sets is searchedfor. Separate and combined analysis using the correlating clonotypesidentified statistically and functionally is performed. The same type ofcharacteristics studied in humans is assessed, for example the level ofthe clonotype, the presence of particular sequence motifs, and thesequence of other related clonotypes. As described for the human study,there is a significant risk of overfilling and hence cross validationtechnique or separate training and testing sets need to be employed.

One utility for the mouse experiment is the availability of cellsallowing for assessment of whether correlating clonotypes are enrichedin a specific subtype of cells. It is studied whether correlatingclonotypes are enriched in some cell subtypes; sequencing from the fullset of lymphocytes and from the specific subtype where correlatingclonotypes are enriched can be done, and this criteria of enrichment canbe used as an extra characteristic to distinguish correlating clonotypesfrom other disease-irrelevant clonotypes. In order to know what cellsubtypes clonotypes are enriched a couple approaches is taken:hypothesis driven and hypothesis free. The first is to try a dozencandidate surface markers on T or B cells in a set of samples. Forexample, one candidate is CD69 on T cells to select activated T cells.For B cells studies have shown the increase of CD27high cells in activeSLE, and therefore that is a good candidate for a marker of cells thatmay have enrichment of the correlating clonotypes. In each of theseexperiments, the specific cell subtypes is purified through FACS. Then asequencing reaction is done for cDNA from the full complement of thelymphocytes as well as for cDNA from the lymphocytes that were purifiedby FACS from a collection of different samples. It is assessed whetherthe two sets of correlating and non-correlating clonotypes are presentin different proportions in the full complement of lymphocyte comparedto the FACS purified subset. Markers that have a large difference can beuseful in identifying correlating clonotypes. Enrichment of clonotypesin subtypes of cells with these markers is used in addition to thesequence parameters to detect correlating clonotypes.

In the hypothesis free approach, markers is searched for which aredifferentially expressed in cells with a correlating clonotype fromother cells. A few cases is chosen where a specific TCR clonotype isclearly correlating with disease, and cases is picked where thatclonotype is highly enriched that it represents the majority of theclonotypes with the same V segment. FACS is done using antibody to thespecific V segment (antibodies against all V segments are commerciallyavailable) to select a population that is highly enriched for cellscarrying the correlating clonotype. The RNA can be prepared from thesecells and the expression of all the genes can be studied by performingan array experiment. As a control, total RNA from lymphocytes can beused and/or RNA from FACS purified cells carrying another irrelevant Vsegment. Markers that maximally distinguish the sample obtained from theFACS purified V segment with the correlating clonotype from the controlscan be searched for. Markers, including surface markers (since it ismuch easier to do FACS with surface proteins) that distinguish the twopopulations can be found. If a consistent RNA marker from samples ofseveral mice is observed it is validated at the protein level. Using thesame samples, antibodies against the marker protein is used in a FACSassay to purify cells carrying the marker protein. More than one markermay be tested to increase the chance of validating one of them. The TCRand/or BCR from the purified cells is sequenced. If the RNA results holdat the protein level then the correlating clonotypes should be enrichedin the purified subset of cells. After validating that RNA results stillhold at the protein level, the results is validated in other samples.Samples that were not subject to the array analysis is subjected to FACSanalysis using the antibody to the marker protein(s). The TCR and/or BCRof the purified cells is sequenced. It is evaluated whether thecorrelating clonotypes are enriched in the cells purified using antibodyto the specific marker(s). This will validate the utility of themarker(s) in the identification of correlating clonotypes.

Example 16 Use of IgH and TCRβ Repertoire to Measure Disease Activity

The algorithm for correlating clonotypes from above can be applied toidentify in all samples of cohorts 1 and 3 correlating clonotypes byvirtue of their sequence and/or markers. Using the level of thecorrelating clonotypes in each patient, an AI score can be generatedthat correlates with a measure of renal function. As described above,there is an overfitting risk and the cross validation technique and/orseparate training and testing set need to be employed. The correlationof AI and renal function measures can be evaluated in a cross sectionalmanner (all study points of all mice). The question of whether the AIscore changes in an individual mouse can also be evaluated when renalfunction changes. This can be evaluated by comparing the AI from highand low renal function in the same animal in a similar manner to what isdescribed in humans.

Example 17 Monitoring for Metastatic Recurrence in Colon Cancer Patients

Many cancers that are detected at a treatable stage still carry anongoing risk to the patient of metastatic tumor recurrence. Suchrecurrences are often detected late and at untreatable stages an can befatal to the patients. One example of such a situation is that ofrecurrent colon cancer. Despite increasingly aggressive colon cancerscreening programs, colon cancer represents one of the most commonmalignancies in the US. Approximately 150,000 patients per year arediagnosed with colon cancer at serious but treatable stages (Stage IIand Stage III). These patients are treated by tumor resection followedby a course of chemotherapy. While these treatments are generallyeffective, there is nonetheless a significant chance that these patientswill have metastatic recurrences of the primary tumor in the yearsfollowing treatment. 50% of Stage III patients for instance will have arecurrence within 5 years of surgery. These recurrences can be eitherisolated (e.g. in the colon or liver) or multifocal. In either case butparticularly if they are isolated, detecting them at an early stage canplay a role in maximizing the chances of successful therapy (surgeryand/or chemotherapy).

There are currently two tests used in post treatment surveillance. CTscan of the abdomen and chest is used to identify tumors visible onthese images. Typically these scans are done at intervals of 6-12 monthsfor the first 5 years post therapy. While these scans can reveal earlystage malignancies, there clinical effectiveness is in debate. Drawbacksof these scans include the fact that they subject the patients tosignificant amounts of radiation which can itself cause further tumorsand the significant expense. Another blood based test has been shown tohave some value: CEA testing. This antibody test measures the level of aprotein in serum that is specific to some colon tumors. The drawback toCEA testing is its lack of sensitivity (<60% of patients with positiveCT scans have a positive CEA test).

In this embodiment of the invention, lymphocytes obtained from theresected primary tumor are used to develop an immune profile that can beused to add sensitivity to a blood based test for early cancerrecurrence TCRs (and/or BCRs) of the lymphocytes found in the resectedtumor can be amplified and sequenced. Clonotypes that are enriched inthe tumor sample are likely relevant to the immune response to thetumor. Subsequent blood draws from the patient can be used to assess thelevel of these clonotypes. A rise in the level of these clonotypes cansignal an immune response to a tumor recurrence. In this case thedetection of the immune response may be more sensitive than thedetection of the tumor marker itself.

Discovery study for the detection of cancer recurrence using acalibration test. A discovery study is performed to determine thelikelihood of detection of recurrence given the profile of blood TCR(and/or BCR). Samples of resected tumor samples as well as follow upblood samples of patients with known outcome are used for this study.TCR (and/or BCR) from all these samples is sequenced. Candidates for thecorrelating clonotypes are those that are present in the TCR (and/orBCR) data from the tumor samples Given the known outcomes in thistraining study one using the standard cross validation techniques, amodel that generates a score (Recurrence Risk) given the level of thedifferent clonotypes is devised. This Recurrence score is thus becalculated in a new patient by measuring the clonotypes in the resectedtumor (calibration point) and the data from the clonotypes found in thesame patient's blood at a later time during the surveillance forrecurrence. The use of the tumor data allows great reduction in thenumber of clonotypes present in blood that are considered in thisanalysis.

Discovery study for the detection of cancer recurrence using acalibration test and a population study. It is likely that not allclonotypes that are enriched in the tumor specimen are relevant to theimmune response to the tumor. There might be some lymphocyte thatexpanded locally due to a favorable inflammatory condition. In anotherembodiment of this invention the discovery study is done using the samesamples but the study is used to identify parameters that distinguish“correlating” from “non-correlating” clonotypes. These parameters caninclude 1) Sequence motif: The motif can be a specific V or J region, acombination VJ, or short sequences in DJ region that is associated witha clonotype being correlating; 2) Size of the clonotype; 3) Level:Absolute level (number of reads per million) or rank level; 4)Similarity to other clonotypes: the presence of other highly relatedclonotypes, like those with silent changes (nucleotide differences thatcode for same amino acids) or those with conservative amino acidchanges; 5) For the BCRs the level of somatic mutations in the clonotypeand/or the number of distinct clonotypes that differ by somaticmutations from some germ line clonotype. 6) Presence in a cell carryinga specific marker. This study then results in an algorithm that canpredict which clonotypes are likely to be correlating with cancerrecurrence in blood given a specific set of clonotypes present in agiven tumor sample. These clonotypes are then be used to develop a scoreof Recurrence Risk in the same manner as described above.

Discovery study for the detection of cancer recurrence using apopulation study. In another embodiment of this invention, theclonotypes measured in the resected tumor are used to generate a modelthat predicts correlating clonotypes in as yet unseen samples. Thismodel can also be used to generate a Recurrence Risk score in a manneranalogous to that described above. In this model there would be no needto measure the clonotypes in the resected cancer tissue in a new patientundergoing recurrence surveillance but rather the Recurrence Risk couldbe assessed by simply measuring the clonotypes in a given blood sample.

Discovery study for the detection of primary colon cancer using apopulation study. As an extension the detection of primary cancers isachieved using the same methodology. With the primary cancers there isno tumor resected that can be used to enrich for relevant clonotypes.However, even in the presence of tumor resection data the additionalsequence and other parameters need used to identify relevant clonotypesand generate a score for likelihood of cancer detection. Therefore byextension if the algorithm is predictive enough one can detect thecancer from blood (or other bodily fluid) without the data from theresected tumor. In this embodiment of the invention, a discovery studywith blood samples from patients preceding their diagnosis of primarycancer need to be available. In an analogous fashion to the onedescribed above, parameters (sequence and other) can be identified topredict the clonotypes that are correlated to the immune system responseto the tumor. A model can then be used to generate a Cancer Risk scorethat predicts the progression risk to colon cancer. This algorithm canthen be applied to new patient's blood sample to measure the risk ofprimary colon cancer.

Example 18 Monitoring for Rejection in Heart Transplant Patients

Heart transplants are a relatively uncommon procedure as the supply oforgans is very limited. 3,500 heart transplants performed every yearworldwide. Each procedure is very expensive and the organs that are usedare priceless. As a result the patients that receive these organs aretreated extremely proactively. In order to measure the state of theimmune reaction to the donated organ at a time at which interventionswith immunosuppressants can be effective, patients are given periodicheart biopsies to measure inflammation of the organ. Based on thesetests, aggressive courses of immunosuppressants may be given. Theseprocedures have several limitations. As invasive surgical proceduresthey have risks to the patient. Furthermore they are expensive and canonly be done at infrequent intervals. A blood based tests based onprofiling the expression of a panel of 11 test genes (Allomap) have beenshown to be quite sensitive in detecting organ rejection but lackssufficient sensitivity to be used as a replacement for biopsy and isinstead used to decide when to do a biopsy. In one embodiment of thisinvention TCR and/or BCR) profiles are used to assess the state of“rejection” and generate a Rejection Risk score that predicts thelikelihood of rejection in a specific time frame. It is conceived that adiscovery study can be performed to determine the likelihood ofrejection given the profile of blood TCR (and/or BCR). This can be usedin the clinic to inform the immunosuppressive therapies that are beingused.

Discovery of correlating clonotypes using a population study. In thisembodiment of the invention a population of post-transplant patientswith blood samples with known clinical outcome is used. TCR (and/or BCR)from all these samples is sequenced and correlation of individualclonotypes with rejection outcome used to distinguish correlating fromnon-correlating clonotypes. Subsequently, parameters are derived thatdistinguish those two classes of clonotypes. These parameters caninclude 1) Sequence motif: The motif can be a specific V or J region, acombination VJ, or short sequences in DJ region that is associated witha clonotype being correlating; 2) Size of the clonotype; 3) Level:Absolute level (number of reads per million) or rank level; 4)Similarity to other clonotypes: the presence of other highly relatedclonotypes, like those with silent changes (nucleotide differences thatcode for same amino acids) or those with conservative amino acidchanges; 5) For the BCRs the level of somatic mutations in the clonotypeand/or the number of distinct clonotypes that differ by somaticmutations from some germ line clonotype. 6) Presence in a cell carryinga specific marker. An alternative or supplemental method to define thecorrelating and non-correlating clonotype would come if the studysamples have biopsy samples of the graft, particularly if it was inactive rejection. It is expected that at that time there is greatenrichment of the correlating clonotypes. Parameters to distinguishthese from the other clonotypes are identified as discussed above.

The profile data from the blood samples is then used to predict thelikelihood of rejection. Given the known outcomes in this training studyone can devise, a model using the standard cross validation techniquesthat generates a Rejection Risk score given the level of the differentclonotypes. Given the profile in a new blood sample of TCR (and/or BCR)at a specific point a Rejection Risk score relating to the likelihood ofrejection can be generated

Discovery of correlating clonotypes using a calibration test. In anotherembodiment a method of identifying correlating clonotypes areimplemented using a calibration test for each patient. This methodinvolves a first biopsy sample be taken post transplant. The presence ofbiopsy material of the graft post transplant offers the possibility ofanalyzing TCRs from the biopsy sample to identify the correlatingclonotypes as defined by those that are prevalent in this sample. Thisset of clonotypessis then followed in blood and a score is generated forthe likelihood of rejection. The algorithm to generate the RejectionRisk score is derived through a discovery study that is similar to theone described above that utilizes the available clinical data and thelevels of the correlating clonotypes to generate a Rejection Risk scorethat approximates the likelihood of rejection.

In this embodiment a specific calibration test is done using materialfrom a first biopsy post transplant but further biopsies could bereplaced by the use of blood samples whose clonotypes could be usedalong with this calibration test to measure a Rejection Risk score.

In addition to the graft biopsy, the blood samples before transplantserve as another calibration point. Clonotypes that are prevalent inthis sample are unlikely to be related to the rejection representingrather the history of prior antigens the patient has seen. Thereforewhen considering the blood samples after transplant one can subtract theclonotypes that were present before the transplant in determining thecorrelating clonotypes. These clonotypes are then used to generate amodel of Rejection Risk.

In this embodiment, two calibration tests would be can be used: oneprior to transplant and one from a biopsy after transplant. Thesecalibrations could then be used along with clonotypes derived from ablood test to measure Rejection Risk.

Discovery of correlating clonotypes using a calibration test and apopulation study. In another embodiment, the identification of thecorrelating clonotypes can be achieved through a combination of theabove approaches. Specifically this can be achieved by using thepopulation study to generate an algorithm to predict correlatingclonotypes. In addition it can be achieved through calibration data fromthe same patient using graft biopsy and/or blood samples pre-transplant.A more preferred embodiment will employ both approaches:population-built algorithm and individual calibration to most accuratelyidentify the correlating clonotypes. A Rejection Risk score is thengenerated using the level of these clonotypes to predict the likelihoodof rejection through the use of the population study as a training set.

In this embodiment, two calibration tests can be used: one prior totransplant and one from a biopsy after transplant. These calibrationscould then be used along with clonotypes derived from a blood test tomeasure Rejection Risk.

The prediction of GVHD can be done in a very similar manner with thesame concept of the population study to generate an algorithm to predictcorrelating clonotypes. Also the “negative” calibration can be generatedfrom the donor sample pre-transplantation. An approach using both thealgorithm and calibration is likely to be more predictive of thecorrelating clonotypes. An algorithm to compute a score of thelikelihood of GVHD given the level of the correlating clonotypes can begenerated using a population study in a manner as described above. Thisalgorithm can then be used for the prediction of the likelihood of GVHDin the next set of patients.

Example 19 Monitoring for PML Infection in MS Patients Treated withNatalizumab

One embodiment of the invention uses TCR and/or BCR profile to detectsubclinical Progressive Multifocal Leukoencephalopathy (PML) in MSpatients. PML is a serious and often fatal disease that causes oftenrapidly progressive demyelinating disease through killingoligodendrocytes that synthesize myelin. It is caused by JC virus thatis present in a latent phase in the majority of the population. In afraction of the immunosuppressed population (e.g., AIDS) the virus isreactivated leading to the development of this serious disease. Inaddition some patients who are being immunosuppressed through the use ofmedication like post transplant patients can also develop PML. Somespecific medication has been linked to the risk of PML in specificpatient populations. For example natalizumab (Tysabri) was associatedwith the development of more than 10 cases of PML among patients withmultiple sclerosis (MS) leading to its withdrawal of the market for aperiod of time. Natalizumab is well accepted to be more effective thanthe other FDA approved medications for multiple sclerosis, but its usehas been limited by the fear of PML development. Once PML is suspected,plasmapheresis can be performed to reduce the concentration of the drugin the patient. The overlap between symptoms of MS and PML can sometimesdelay the detection of PML. Early detection of subclinical PML isurgently needed.

These clonotypes may be discerned from blood samples from a populationwhere some patients developed PML. This population can be used toidentify clonotypes that correlate with the later development of PML.With the availability of these clonotypes an algorithm to identifyparameters that distinguish these from other clonotypes can begenerated.

Discovery of correlating clonotypes using a population study. In thiscase an algorithm is generated to predict the clonotypes that arerelevant to the emergence of PML. The algorithm can be trained on a setof clonotypes deemed to be correlating with the disease. In thisembodiment of the invention blood (or other body fluid) samples in adiscovery study from a population of patients with a latent infectionwith JC virus some of whom go on to develop PML can be used. TCR (and/orBCR) from all these samples can be sequenced and correlation ofindividual clonotypes with infectious agent reactivation outcome can beused to distinguish correlating from non-correlating clonotypes.Parameters that distinguish those two classes of clonotypes can beidentified. These parameters can include 1) Sequence motif: The motifcan be a specific V or J region, a combination VJ, or short sequences inDJ region that is associated with a clonotype being correlating; 2) Sizeof the clonotype; 3) Level: Absolute level (number of reads per million)or rank level; 4) Similarity to other clonotypes: the presence of otherhighly related clonotypes, like those with silent changes (nucleotidedifferences that code for same amino acids) or those with conservativeamino acid changes; 5) For the BCRs the level of somatic mutations inthe clonotype and/or the number of distinct clonotypes that differ bysomatic mutations from some germ line clonotype. 6) Presence in a cellcarrying a specific marker. An alternative or supplemental method todefine the correlating and non-correlating clonotype would come from aset of patients who are mounting an immune response to the sameinfectious agent. Enriched clonotypes (particularly those that are at asignificantly higher level than before the immune response) in thesepatients can be considered correlating and parameters that distinguishthem from other clonotypes can be identified.

Similarly the correlating clonotypes can be identified from samples ofpatients with active PML or from in vitro studies to identify clonotypesthat respond to JC virus antigen. The responding clonotypes mayoriginate from one or a plurality of subjects that may be healthy orinfected with the infectious agent. These clonotypes can be consideredcorrelating and parameters that distinguish them from other clonotypescan be identified. The profile data from the samples in the discoverystudy is then used to predict the likelihood of reactivation. Given theknown outcomes in this training study one can devise using the standardcross validation techniques, a model that generates a PML Risk scoregiven the level of the different clonotypes. So given the profile in ablood sample of TCR (and/or BCR) at a specific point a score relating tothe likelihood of reactivation can be generated. This algorithm can nowbe used with data from a novel patient to predict the patient'scorrelating clonotypes as well as to generate a PML Risk score for thelikelihood of reactivation.

In a very similar manner other infection-related outcomes can bestudied. For example in addition to reactivation of latent infection,one can assess clearance of infection. Furthermore given the TCR and/orBCR repertoire one may be able to evaluate likelihood of having immunityfor a specific infectious agent

Example 20 Monitoring for Reactivation of Latent Infections

In another embodiment TCR and BCR profiling can be used to monitorinfections that have periods of acute infection followed by latency andreactivation. Examples of such diseases include Hepatitis B and C aswell as Herpes viruses. Predicting infections at early stage would bedesirable.

Discovery of correlating clonotypes using a calibration test. In anotherembodiment a method of identifying correlating clonotypes can beimplemented using a calibration test for each patient. The presence of abiological sample from the same patient at a previous time point whenthe patient was mounting an immune response to the infectious agent canserve to identify the correlating clonotypes. This set of clonotypes canthen be followed in blood and a Reactivation Risk score is generated forthe likelihood of reactivation. The algorithm to generate the score isderived through a discovery study that is similar to the one describedabove that utilizes the available clinical data and the counts of thecorrelating clonotypes to generate a Reactivation Risk score thatapproximates the likelihood of reactivation. To use this score a sampletaken from a new patient in clinical practice during a period of acuteinfection. This data would be used along with a subsequent sample takenduring the latent period to measure the Reactivation Risk for clinicalpurposes.

Discovery of correlating clonotypes using a calibration test and apopulation study. In another embodiment, the identification of thecorrelating clonotypes can be achieved through a combination of theabove approaches. Specifically this can be achieved by using thepopulation study to generate an algorithm to predict correlatingclonotypes. The correlating clonotypes can be obtained from a populationstudy of patients with known outcome of the infection and/or a set ofpatients with active immune response to the infectious agent, and/orfrom in vitro experiments to identify clonotypes reactive with theinfectious agent. In addition it can be achieved through calibrationdata from the same patient using older data points at the time of anactive immune response against the relevant infectious agent. A morepreferred embodiment will employ both approaches: population-builtalgorithm and individual calibration to most accurately identify thecorrelating clonotypes. A Reactivation Risk score is then generatedusing the level of these clonotypes to predict the likelihood ofreactivation through the use of the population study as a training set.To use this score a sample taken from a new patient in the clinic duringa period of acute infection is profiled. This data would be used alongwith a subsequent sample taken during the latent period to measure theReactivation Risk for clinical purposes. A similar structure can beemployed to study infectious agent clearance and or immunity to it.

Example 21 Monitoring for Allergic Response During Immunotherapy

Allergic rhinitis is a common condition afflicting ˜11% of the USpopulation. This is typically an allergy to pollen or dust. Eliminatingthe exposure is difficult and it involves vigilant effort. The mostcommon treatments used in chronic rhinitis are decongestants,antihistamines, and nasal steroids. In severe cases immunotherapy isdone. The goal of the immunotherapy is to de-sensitize the patient.First a challenge with many potential allergens is done to identify thespecific allergen the patient is reacting to. Then the patient isinjected with increasing amount of allergen over a period of months toyears until a maintenance dose is achieved, and the treatment is thencontinued for several years. Typically the patient can feel animprovement in symptoms within 3-6 months, but that can also be as lateas 12-18 months, but a large fraction of the patients do not benefitfrom the treatment or have relapses. One reason for the slow doseescalation is the risk of anaphylaxis if the patient is given a highdose of allergen before s/he sufficiently de-sensitized.

In one embodiment of this invention TCR (and/or BCR) profiles are usedto assess the state of disease in allergic rhinitis and generate anAllergy Score that predicts how prone the patient to mount an allergicresponse should s/he be exposed to the relevant allergen. It isconceived that a discovery study can be performed to determine thelikelihood of allergy response given the profile of blood TCR (and/orBCR). This can be used in tailoring the immunotherapy treatment.Possible clinical decision can be to discontinue the treatment if it isdeemed ineffective, continue the injection regimen, or accelerate thetreatment to reach the maintenance dose faster.

Discovery of correlating clonotypes using a population study. In thisembodiment of the invention a population of allergic rhinitis patientson immunotherapy with blood samples with known clinical outcome can beused. TCR (and/or BCR) from all these samples can be sequenced andcorrelation of individual clonotypes with allergy outcome can be used todistinguish correlating from non-correlating clonotypes. Subsequently,parameters can be derived that distinguish those two classes ofclonotypes. These parameters can include 1) Sequence motif: The motifcan be a specific V or J region, a combination VJ, or short sequences inDJ region that is associated with a clonotype being correlating; 2) Sizeof the clonotype; 3) Level: Absolute level (number of reads per million)or rank level; 4) Similarity to other clonotypes: the presence of otherhighly related clonotypes, like those with silent changes (nucleotidedifferences that code for same amino acids) or those with conservativeamino acid changes; 5) For the BCRs the level of somatic mutations inthe clonotype and/or the number of distinct clonotypes that differ bysomatic mutations from some germ line clonotype. 6) Presence in a cellcarrying a specific marker. An alternative or supplemental method todefine the correlating and non-correlating clonotype would use biopsy ofpositive allergy test material from patients positive for a specificallergen. At the site of injection of the allergen it is expected thatthere is great enrichment of the correlating clonotypes. Parameters todistinguish these from the other clonotypes can be identified asdiscussed previously.

The profile data from the blood samples is then used to predict theallergy state. Given the known outcomes in this training study one candevise, a model using the standard cross validation techniques thatgenerates an Allergy Score given the level of the different clonotypes.Given the profile in a new blood sample of TCR (and/or BCR) at aspecific point, an Allergy Score can be generated to estimate the degreeto which this patient is prone to mount an allergic response.

Discovery of correlating clonotypes using a calibration test. In anotherembodiment a method of identifying correlating clonotypes can beimplemented using a calibration test for each patient. This methodinvolves a biopsy sample from a site with a positive allergen responsebe taken from the patient. This can be from the initial allergy testthat was performed to determine the specific allergen the patient isresponding to or sample from the site of any further treatmentinjections. This can be done more than once to ensure that theappropriate clonotypes are being followed in case there is some epitopespreading. TCR and/or BCR from these biopsy samples can be used toidentify the correlating clonotypes as defined by those that areprevalent in this sample. This set of clonotypes can then be followed inblood and a score is generated for the likelihood of allergy response.The algorithm to generate the Allergy Score is derived through adiscovery study that is similar to the one described above that utilizesthe available clinical data and the levels of the correlating clonotypesto generate an Allergy Score that estimates the allergy state.

Discovery of correlating clonotypes using a calibration test and apopulation study. In another embodiment, the identification of thecorrelating clonotypes can be achieved through a combination of theabove approaches. Specifically this can be achieved by using thepopulation study to generate an algorithm to predict correlatingclonotypes. In addition it can be achieved through calibration data fromthe same patient using biopsy from a site with a positive allergenresponse. A more preferred embodiment will employ both approaches:population-built algorithm and individual calibration to most accuratelyidentify the correlating clonotypes. An Allergy Score is then generatedusing the level of these clonotypes to predict the state of allergythrough the use of the population study as a training set.

Example 22 Amplification of IgH Sequences from Genomic DNA

In this example, amplification of IgH sequences from genomic DNA isdescribed. Such amplification is advantageous because (1) the level of aclonotype in genomic DNA can be readily converted to number of cells,and (2) in some lymphoid neoplasms, RNA may not be expressed for therelevant immune receptor rearrangement.

Amplification of immune receptor rearrangement is important for thedetection of lymphoid neoplasms. B cell neoplasms are more common than Tcell tumors and IgH is the most common rearranged immune receptor in Bcell neoplasms. Because of somatic hypermutation, reliability ofamplifying of IgH from genomic DNA may be increase by amplifying withmultiple primers for each V segment, although there is a risk ofdifferential amplification. In amplification from genomic DNA, the sameV primers were used that were used in amplification from cDNA. Each Vsegment is amplified by 3 primers (in 3 distinct regions of the Vsegment: A, B, and C) in three different reactions (Tables 5-7,respectively) (see FIG. 4A).

TABLE 5  Human IgH V Segment Primers for Reaction A(All the primers have a common 14 bp (AGATCGGAAGAGCA) (SEQ ID NO 165)appended to their 5′ end) Sequence SEQ ID NO TCGCAGACCCTCTCACTCACCTGTG62 GATCTCCTGTAAGGGTTCTGGATACAGCT 61 AAACCCACACAGACCCTCACGCTGAC 44AAACCCACAGAGACCCTCACGCTGAC 45 AAACCCACACAGACCCTCACACTGAC 46CCTCAGTGAAGGTCTCCTGCAAGG 40 CCTCGGTGAAGGTCTCCTGCAAGG 41CCTCAGTGAAGGTTTCCTGCAAGG 42 GGGCTACAGTGAAAATCTCCTGCAAGG 43CTTCGGAGACCCTGTCCCTCACCTG 56 CTTCGGACACCCTGTCCCTCACCTG 57CTTCACAGACCCTGTCCCTCACCTG 58 CTTCGGAGACCCCGTCCCTCACCTG 59CGGGGACCCTGTCCCTCACCTG 60 CTGGGGGGTCCCTGAGACTCTCCTG 47CTGGGGGGTCCCTTAGACTCTCCTG 48 CAGGGCGGTCCCTGAGACTCTCCTG 49CAGGGCCGTCCCTGAGACTCTCCTG 50 CTGGGGGGTCCCTGAAACTCTCCTG 51CTGGCAGGTCCCTGAGACTCTCCTG 52 CTGGAGGGTCCCTGAGACTCTCCTG 53CTGGGAGGTCCCTGAGACTCTCCTG 54 TGGGGGGGCCCTGAGACTCTCCT 55

TABLE 6  Human IgH V Segment Primers for Reaction B(All the primers have a common 14 bp (AGATCGGAAGAGCA) (SEQ ID NO 165)appended to their 5′ end) Sequence SEQ ID NO TGGATCAGGCAGTCCCCATCGAGAG63 GCTGGGTGCGCCAGATGCCC 64 GTGTGAGCTGGATCCGTCAGCC 131GTGTGGGCTGGATCCGTCAGCC 132 GTGCGACAGGCCCCTGGACAA 67GGGTGCGACAGGCCACTGGACAA 68 GTGCGCCAGGCCCCCGGACAA 69GGGTGCGACAGGCTCGTGGACAA 70 GGGTGCAACAGGCCCCTGGAAAA 71GGGTGCGACAGGCTCCTGGAAAA 72 GTGCGACAGGCCCCCGGACAA 73GTGCGACAGGCCCCCAGACAA 74 TCCGCCAGCCCCCAGGGAAGG 75 TCCGGCAGCCCCCAGGGAAGG76 TCCGGCAGCCACCAGGGAAGG 77 TCCGCCAGCACCCAGGGAAGG 78 TCCGGCAGCCCGCCGGGAA79 TCCGGCAGCCGCCGGGGAA 80 TCCGGCAGCCCGCTGGGAAGG 81 TCCGCCAGCCCCTAGGGAAGG82 GGTCCGCCAGGCTCCAGGGAA 83 GTTCCGCCAGGCTCCAGGGAA 84GGTCCGCCAGGCTTCCGGGAA 85 GGTCCGTCAAGCTCCGGGGAA 86 GATCCGCCAGGCTCCAGGGAA87 GGTCCGCCAAGCTCCAGGGAA 88 GGTCCGCCAGGCTCCAGGCAA 89GGTCCGCCAGGCCCCAGGCAA 90 GGTCCGCCAGGCTCCGGGCAA 91GGGTCCGTCAAGCTCCAGGGAAGG 92 CTGGGTCCGCCAAGCTACAGGAAA 93GGTCCGCCAGCCTCCAGGGAA 94 GGTCCGGCAAGCTCCAGGGAA 95 GTGCGAGCTGGATCCGTCAGCC133

TABLE 7  Human IgH V Segment Primers for Reaction C(All the primers have a common 14 bp (AGATCGGAAGAGCA) (SEQ ID NO 165)appended to their 5′ end) Sequence SEQ ID NO GCAGCCTAAAGGCTGAGGACACTG134 CTCTGTGACTCCCGAGGACACGGCT 97 AGTGGAGCAGCCTGAAGGCCTC 98TGACCAACATGGACCCTGTGGACAC 99 ACATGGAGCTGAGCAGCCTGAGATC 100ACATGGAGCTGAGCAGGCTGAGATC 101 ACATGGAGCTGAGGAGCCTGAGATC 102ACATGGAGCTGAGGAGCCTAAGATCTGA 103 GAGCTCTGTGACCGCCGCGGAC 104GAGCTCTGTGACCGCCGTGGACA 105 GAGCTCTGTGACCGCTGCAGACACG 106GAGCTCTGTGACCGCTGCGGACA 107 GAGCTCTGTGACTGCCGCAGACACG 108GAGCTCTGTGACTGCAGCAGACACG 109 GAGCTCTGTGACTGCCGCGGACA 110GAGCTCTGTGACCGCGGACGCG 111 GGCTCTGTGACCGCCGCGGAC 112GAGCTCTGTGACCGCCGCAGACA 113 GAGCTCTGTGACCGCTGACACGG 114CAAATGAACAGCCTGAGAGCCGAGGACA 115 CAAATGAACAGCCTGAAAACCGAGGACA 116CAAATGAACAGTCTGAAAACCGAGGACA 117 CAAATGATCAGCCTGAAAACCGAGGACA 118CAAATGAACAGTCTGAGAACTGAGGACACC 119 CAAATGAACAGTCTGAGAGCCGAGGACA 120CAAATGAACAGCCTGAGAGCTGAGGACA 121 CAAATGAGCAGCCTGAGAGCTGAGGACA 122CAAATGAACAGCCTGAGAGACGAGGACA 123 CAAATGGGCAGCCTGAGAGCTGAGGACA 124CAAATGAACAGCCTGAGAGCCGGGGA 125 CAAATGAACAGTCTGAGAGCTGAGGACA 126CAAATGAGCAGTCTGAGAGCTGAGGACA 127 GCACGCTAAAGGCTGAGGACACTG 135

Amplification of IgH from genomic DNA has several differences from itsamplification from cDNA. The C segment gets attached to the VDJ regionthrough splicing and hence sequences of the C segment can be used foramplification from cDNA but not genomic DNA. The use of C segment allowsthe use of two distinct primers in the 1^(st) and 2^(nd) amplificationsincreasing the specificity. For the amplification from genomic DNA wehave opted to use primers that are complementary to the J sequences(Table 8).

TABLE 8  Human IgH J Segment Primers* SEQ J Segment Primer ID NOACGAGCCTCATGCGTAGANct acCTGAGGAGACGGTGACC 136ACGAGCCTCATGCGTAGANct acCTGAGGAGAC GTGACC 137ACGAGCCTCATGCGTAGANcttacCTGA GAGACGGTGACC 138ACGAGCCTCATGCGTAGANcttacCTGAGGAGACGGTGACC 139 *The J segment primersused. The 18 bp on the 5′ are common sequences that are appended to thesequence complementary to the J segment in order to allow the secondstage amplification. The position N signifies one random position inorder to obtain diversity in the sequenced clusters. The small lettersequence are in the intron, and the capital letter sequences in the 3′of the sequence are in the exon. The letters in italics emphasize thebases that differ among the primers.

These primers span the exon-intron boundaries, and the four primersutilized amplify the different J segments and alleles described in theIMGT database. Primers of the second stage do not have any sequencescomplementary to genomic sequences.

Using the J primers over constant region primers complementary to theIgG constant region allows the assessment of the other classes (IgM,IgD, IgA, and IgE).

In the case of cDNA there is a choice of whether to use the J primer orthe constant region primers. Several constant region primers can be usedto amplify all the classes and sequence some of the constant regionbefore entering into the J sequence in order to link the information onthe clonotype and its specific class. The sequencing reads for many ofthe sequencing technologies are short and would be difficult toaccomplish this. One of the current platforms on the market (454 Roche)does have a longer read but it has lower throughput than otherplatforms. As these technologies develop further this option becomepossible. With the current short reads (<100 bp) our work on the genomicDNA assay suggests that for amplification from cDNA can be done usingboth the J and the C priming approach. We can implement amplificationusing J primers from cDNA. However given that the exonic segment ofthese primers may be too short for specific amplification from cDNA,potentially the first stage PCR can be done using a set of constantregion primers encompassing all the different classes (and the V segmentprimers on the other side as we have demonstrated). Then the secondstage PCR can be done with the J primers which are long enough to havehigh specificity for second stage PCR which is utilizing a lowcomplexity template. The products are then be sequenced. As statedabove, the disadvantage compared with the scheme demonstrated for IgG isthat somatic mutations in the J sequence may inhibit the amplification.The advantage is that all the different classes are assessed, eventhough the information on the class of each clonotype is not fullydetermined. Potentially one can do class specific amplification IgG,IgM, IgD, IgA, or IgE and compare with the overall picture obtained fromusing all the primers followed by J primer. For example one can compareclonotype profile obtained from IgG amplification to that using all theprimers followed by J primer. The difference presumably would be due tosomatic mutation in the J primer (which can be readily identified in thereaction using the IgG primer) and clonotypes of the other classes,which can then be quantified.

The use of J primers in cDNA also allows the direct comparison betweenthe cDNA and genomic DNA results. This would provide expression levelinformation at a clonotype level and can indeed have functionalrelevance. One aspect of this invention is that comparing the clonotypeprofile of cDNA and genomic DNA from the same blood or other biologicalsample identifies clonotypes that have different frequencies indicatingunusually high or low expression per cell. This functional informationcan be utilized to predict whether a clonotype is likely to becorrelating with disease or not. In addition the level of expression percell of a clonotype correlating with a disease can be used to determinethe disease activity or the likelihood of a disease outcome. For exampleobtaining the same level in the cDNA assay for a correlating clonotypein two individuals may still indicate the patients have differentdisease activities if the clonotypes level of expression per cell (asdetermined by comparison with the genomic DNA clonotype profiling) isdifferent.

The second stage PCR is to attach the sequences necessary for theamplification. The primers used in the second stage are listed in Table9.

TABLE 9  Common primers* SEQ ID Primer Sequence Utilization NOAATGATACGGCGACCACCGAGATCT Third stage PCR 140 CAAGCAGAAGACGGCATACGAGATThird stage PCR 37 CAAGCAGAAGACGGCATACGAGATN Common second 141NNNNNGTGACTGGAGTTCAGACGTG stage PCR TGCTCTTCCGATCTTGATGGCTCAAACAAGGAGACCT First stage PCR 142 for mouse T RβAATGATACGGCGACCACCGAGATCT Second stage PCR 143 GACCTTGGGTGGAGTCACATTTCTCfor mouse T Rβ AGATCCT AATGATACGGCGACCACCGAGATCT Second stage for 144ACACTCTTTCCCTACACGAGCCTCA human IgH from TGCGTAGA genomic DNA *Thirdstage is an optional amplification stage for all the assays (e.g, mouseTCRβ and human IgH). It is done to ensure the integrity of the endsequences that hybridize to the oligonucleotides attached to the flowcell. The common second stage primer is used in all the assays (e.g,mouse TCRβ and human IgH). Note that use of N in the common second stageprimer is to denote the fact that each of these primers contains aunique 6 base pair tag to allow samples to be later identified.

Amplification is possible using the above primers and other sequencesthat are substantially similar. FIGS. 8A-8B show examples of suchamplifications, which were successful at least in the range of genomicDNA 50-2,000 μg in 20 μl of input genomic DNA.

The assay needs to accommodate a large dynamic range of DNA. Biopsysamples may not have large amount of material, but given that the tumoris likely to be greatly enriched there is no need for a large amount ofstarting material. On the other hand, one million cells will have ˜6 μgof genomic DNA. PBMC containing 1 million B cells will likely have ˜20μg of genomic DNA. To be able to assess 1 million B cells, ˜6.6 μg ofgenomic DNA is used in each of the 3 PCR reactions. Of note is that ifthere is a somatic mutation in the sequence complementary to one of theprimers then in this example only ˜660K B cells are being interrogated.It is useful if the assay works over the range of 50 to 10,000 ng. Theassay has been demonstrated to work in the range of 50-2,000 ng of DNAin 20 μl. By scaling up the reaction to 100 μl, 10 μg of DNA can beused.

Example 23 Monitoring Acute Lymphoblastic Leukemia (ALL)

Minimal Residual Disease (MRD) is an important prognostic factor for thestratification of childhood ALL. MRD is typically tested in the bonemarrow in the few weeks after induction therapy. More sensitivedetection of leukemic cells can allow the monitoring for cancerrecurrence in the blood.

Clonotype profiling to assess the level of the tumor clonotype in theblood is used to detect the leukemic cells sensitively.

The calibration is identified through the interrogation of a sample withhigh leukemic load. Leukemic cells typically are present at highfrequency in diagnostic samples (blood or bone marrow). The diagnosticsample is often sequenced for several rearrangements.

If the tumor is B cell, the fully rearranged IgH, the partial D-Jrearranged IgH, IgK including the Kde rearrangements can be assessed.

Cross lineage rearrangements frequently occur, and the most frequent isthe partially rearranged (V-D) or (D-D) TCRβ. For T cells frequentrearrangement occur for TCRα and TCRβ, and at lower frequency TCRα.Sequencing the clonotype repertoire for these different rearrangementsidentifies the particular tumor the rearrangement present in the tumor.The blood level of the specific sequence can then be monitored.

The monitoring test may involve the relevant rearrangements types only.For example if the tumor rearrangements identified in the diagnosticsample are IgH and IgK, then IgH and IgK would be amplified andsequenced in later blood samples. DNA from up to about 1 million B cellsfrom these samples can be used to amplify IgH and IgK and up to about 1million or more sequencing reads can be obtained, which gives an assaysensitivity of 1 in a million B cell is ˜ to a sensitivity of 1 in 10million white blood cells. With this great sensitivity, leukemic cellsare likely to be detected significantly before a frank relapse.

Clonal evolution has been described in ALL. This can occur through Vreplacement or other mechanisms. To detect evolution, we will identifyclonotypes that are related to those present in the diagnostic sample.For example, clonotypes with the same D-J junction, but with a differentV will be identified. The presence of these related clonotypes atappreciable frequency in the diagnostic sample increases the likelihoodof the relevance of the clonotype. Following more than one rearrangement(for this example tumor IgH and IgK) can also ameliorate this problem.

The mere presence of leukemic cells may not necessarily be sufficientfor predicting relapse. A discovery study is performed with longitudinalblood samples for patients with known outcome. We will assess in thesesamples whether the mere presence of leukemic clonotype is sufficient topredict a relapse some time later. In addition we will assess the changein the frequency of these clonotypes as a predictor of relapse. Inaddition to the clonotype frequency, markers on the leukemic cells maybe indicative of relapse. Sequencing can be performed before and afterthe enrichment of cells with the relevant marker. Therefore the totalfrequency of the clonotype is determined. In addition the fraction ofthese cells with the relevant marker is determined allowing a moreprecise estimation of the risk of relapse.

In accordance with the above, in one aspect the invention providesassays for MRD based on clonotype profile measurements. Such assaysinclude using clonotype profiles to (i) monitor the presence andabundance of patient-specific clonotypes associated with the disease,including evolved clones, (ii) provide counts of lymphocytes from whichthe clonotypes are derived, and (iii) provide a measure of clonality(that is, a measure of how “skewed” a profile is to one or a fewclonotypes). In one aspect, an assay is provided that gives values for(i) and (ii) simultaneously; and in another aspect, an assay is providedthat gives all three quantities simultaneously from a single measurementof a patient's clonotype profile.

Example 24 Monitoring of Transplant Rejection of a Solid Organ

Rejection of a solid organ transplant can occur through two distinctpathways: direct and indirect presentation. The direct pathway uses thedonor antigen presenting cells that are transferred with the transplant.T cell receptors are recognizing in this case the donor HLA. Theindirect pathway on the other hand, occurs some time later. In this casethe donor peptides are presented by the recipient HLA to T cells.

,Samples from biopsy of the transplanted organ can be used to identifyrelevant sequences for calibration of the relevant T (or B) cellreceptors in transplant rejection. Clonotypes enriched in a biopsy ofthe transplanted organ can be compared to the blood at the time ofrejection to identify clonotypes that are relevant to the rejection. Thelevel of these clonotypes in the blood are then monitored to predict thestate of the rejection.

Antigen specific calibration is also performed. To identify clonotypesrelevant to rejection by the direct pathway, donor lymphocytes areirradiated and mixed with recipient PBMC. Recipient PBMC able torecognize donor lymphocytes are activated. Isolation of these activatedcells is done by any of several techniques. For example these cells areisolated by virtue of cytokine release by intracellular cytokinestaining or cytokine capture techniques. Instead (or in addition) toisolation the cells are allowed to replicate in vitro. Comparingsequences of the isolated (and/or the replicated cells) with thepre-activation sequences identifies clonotypes that interact with thedonor PBMC. The same procedures of T cell activation without theaddition of antigen followed by sequencing in order to subtractpotential background of antigen-independent activation is alsoperformed. The blood level of the transplant rejection relevantclonotypes is then monitored to assess the rejection activity of thedirect pathway. A measure of the overall diversity is then used tomonitor the level of rejection by the direct pathway.

To identify clonotypes relevant to rejection by the indirect pathway,donor antigens need to be presented in the context of the recipient HLA.Since the donor HLA is often an important antigen, the donor HLA isincubated with recipient antigen presenting cells that are able topresent peptides from the donor HLA in the context of the recipient HLAto recipient T cells. In a manner similar to what is described above,these cells are isolated and replicated to identify clonotypes that areinteracting with the donor HLA in the indirect pathway. Alternativelythe same procedures of T cell activation without the addition of antigenfollowed by sequencing in order to subtract potential background ofantigen-independent activation is performed. Alternatively donor cellsand not just HLA are used as a source of antigen. The donor cells areprepared in a manner that makes it easy for recipient antigen presentingcells to present donor antigen in the context of recipient HLA. This isdone by several alternative methods including lysing using severalcycles of freezing and thawing or by sonication before addition to theantigen recipient presenting cells. The clonotypes that are activated bythese antigens in the context of recipient HLA, are then identified bysequencing the isolated and/or replicated cells as described above Thesame procedures of T cell activation without the addition of antigenfollowed by sequencing in order to subtract potential background ofantigen-independent activation is alternatively performed. Once theseclonotypes are identified their blood level is monitored to assess therejection activity of the indirect pathway.

Example 25 Cancer Recurrence

Cancer recurrence is detected by the detection of the immune response tothe tumor. The level of T and B cell clonotypes that are relevant to thetumor are used to detect cancer recurrence. The increase in the bloodlevel of the relevant T and B cells (or the frequency of the relevantclonotypes cDNA obtained in blood) is detected and indicates recognitionby the immune system of tumor recurrence.

The reduction of these levels is also detected and indicates the successof the tumor in evading the immune system and hence the development ofcancer recurrence.

Cell markers change on the cells containing relevant clonotypes aredetected and indicate tumor recurrence in the absence of a change in thefrequency of the relevant clonotypes. These latter cases can be areflection of tumor effect on the immune cells to make them ineffectiveor anergic.

In order to determine the relevant clonotypes in an individual, samplesof the original tumor as well as algorithms developed in populationstudies are used. Alternatively, tumor-specific antigen are used todefine clonotypes that interact with the tumor cells. For example B or Tcells that interact with some tumor-specific antigens are captured andsequenced before and after this enrichment to determine the specificclonotypes that interact with the specific antigens.

In vitro experimentation are performed using techniques like the onesdiscussed above (e.g, tetramer binding, intracellular cytokine staining,or cytokine capture) to determine in a patient's sample the specificclonotypes interacting with the particular tumor-specific antigens. Oncethese clonotypes are defined, their level is monitored in other bloodsamples. Change in the level of these clonotypes indicates a tumorrecurrence.

Cancer relevant clonotypes in an individual are identified using an invitro assay to determine clonotypes interacting with the tumor. Tumorcells are lysed using repeated cycles of freezing and thawing orsonication. This preparation is added to autologous antigen presentingcells (or to autologous PBMC containing antigen presenting cells and Tcells). The mixture is added to autologous T cells and clonotypes thatare activated by antigen are identified by sequencing isolated and/orreplicated T cells and comparing to the sequence of the unenrichedmaterial as described above. The same procedure of T cell activation isperformed without the addition of antigen followed by sequencing inorder to optionally subtract potential background of antigen-independentactivation. Once the relevant clonotypes are determined their level inthe blood is monitored in order to assess the likelihood of recurrence.

Where the cancer is of cells that are capable of antigen presentation,the tumor cells may optionally not be lysed as they may serve as theantigen presenting cells. Lymphoma, a B cell tumor that may be able topresent antigens in some cases, serves as an antigen presenting cell.The tumor cells are optionally activated in vitro to improve its antigenpresenting capability. These tumors are then mixed with autologous Tcells (or PBMC). Sequencing of the T cell clonotypes before and afterenrichment identifies the cancer-relevant clonotypes. The level of theseclonotypes is then monitored in the blood to determine the risk ofrecurrence.

The level of the clonotype in a specific type of cells, e.g., those withspecific surface markers is monitored to detect cancer cells which mayevade the immune system. So for two patients with the same level of therelevant clonotype, then depending on the markers contained in the cellcontaining the clonotypes, one patient may have a higher likelihood ofrecurrence compared to another. In order to obtain the information,sequencing can be done before and after enrichment of cells by theparticular marker. Therefore the total as well as the fraction andnumber of clonotype cells with the particular marker can be measured.

Example 26 Monitoring of Hepatitis C Infection

The acute infection of hepatitis C is often accompanied by an immuneresponse that is capable of clearing the infection in ˜15% of cases. Theability to clear the infection has been shown to be associated withcertain HLA genotypes. In the majority of cases the virus is not clearedand a chronic infection occurs. During this chronic infection the virusis able to evade the immune response which is probably responsible tothe much of the resulting liver damage. The most effective treatment forthe disease is interferon. This treatment kills the virus at leastpartly through activation of the immune response. Monitoring of theimmune response can therefore be helpful in different states during thecourse of the disease. During the acute phase assessing the extent ofthe immune reaction may be helpful in predicting who is likely to clearthe virus. During the chronic phase measurement of the level of theimmune response can provide an indication of the degree of the liverinflammation. Finally, the evaluation of the immune reaction duringinterferon treatment can provide an early indication of whethertreatment is being effective. The assessment of the immune response canbe done by measurement of the T and B cell repertoire by sequencing asdescribed above.

The identification of the clonotypes relevant to hepatitis C in eachindividual is done by several methods. Hepatitis C antigens are used asindividual peptides, a mixture of peptides, proteins, or the full virus.T cell and/or B cells interacting with antigen are identified byevidence of enrichment of the clonotypes in cells activated by antigenscompared to their level in the rest of the cells. In addition liverbiopsy is optionally done for these patients during the course oftreatment. That provides additional or alternative means to identifyhepatitis C relevant clonotypes. Clonotypes significantly more enrichedin the liver compared to the blood are likely to be relevant at leastregarding the inflammatory process in the liver. Therefore their levelsin the blood in later points are monitored to assess the liverinflammatory activity. Finally discovery studies in a population ofpatients may indicate a set of sequences or motifs that are relevant tohepatitis C. In this population study relevant clonotypes are identifiedby the virtue of their correlation with disease or enrichment in liverbiopsy and algorithms to distinguish these clonotypes from others isdiscovered. Some of criteria include clonotype frequency, rank, sequencesimilarity of multiple clonotypes, or sequence motif as well as thepresence of some cell marker.

HLA typing is used as a stratification method. Specific motifs arepredictive only in the context of specific HLA types.

Virus infected cells can sometimes evade the immune system. Thereforemonitoring of the level of the clonotype in a specific type of cells,e.g., those with specific surface markers, is performed. So for twopatients with the same level of the relevant clonotype, then dependingon the markers contained in the cell containing the clonotypes, onepatient is mounting a more rigorous response than the other. Forexample, the effect of interferon treatment may be in the qualitative aswell as quantitative change in the clonotype cells. Therefore it isimportant to obtain the level of the clonotype and define whether theyhave specific cell markers. In order to obtain the information,sequencing is done before and after enrichment of cells by theparticular marker. Therefore the total as well as the fraction andnumber of clonotype cells with the particular marker can be measured.

Example 27 Drug Hypersensitivity

The identification of clonotypes that are relevant to a specific drughypersensitivity is done using population studies. In these studiesclonotypes that correlate with the ADR are identified andcharacteristics that distinguish them from the other clonotypes areidentified by different criteria like frequency, rank, relative changebefore and after treatment, sequence similarity of multiple clonotypes,sequence motif, as well as the presence of cell marker. Sequence motifcan be HLA-dependent where different motifs are determined to berelevant to different corresponding HLA sequences.

Another method for identification of drug-hypersensitivity relevantclonotypes is by interaction with antigen. Drugs and/or itsmetabolite(s) is used to capture B cells that interact with it.Similarly the drug or its metabolites are incubated with autologousantigen presenting cells optimally before or at the same time ofaddition of T cells. Activated T cells are isolated or replicated usingsome of the methods discussed above to obtain an antigen-enriched cells.These antigen-enriched cells are then sequenced and clonotypes enrichedin these cells compared with the un-enriched cells are identified asrelevant to the drug interaction.

The same procedures of T cell activation without the addition of antigenis also performed followed by sequencing in order to subtract potentialbackground of drug antigen independent activation.

This calibration test is done before or after taking the drug. Bloodsamples before and after taking the drug and the in vivo increase in thelevel of the clonotype are used as an additional criteria to define therelevant clonotypes. Once these clonotypes are identified they aremonitored to predict the likelihood of a drug hypersensitivity. Thisinvention utilizes the lymphocyte activation with drugs merely to definethe relevant clonotypes and the blood level of these clonotypes can bemonitored in subsequent samples by sequencing generating a sensitive andspecific method to predict ADRs. The drugs that are used are smallmolecules or biological, like antibodies. Similarly, a drug metaboliteor a combination of metabolites is used to identify T cells thatinteract with it as described above. The metabolite is generated bychemical synthesis or purified from a biological sample. For example thedrug is introduced into an organism and the drug metabolites arepurified for use in the assay. The metabolites are also obtained byprocessing the drug by cells in vitro.

The level of the clonotype in a specific type of cells, e.g., those withspecific surface markers is monitored The increase in a clonotype levelin cells carrying activation markers after the introduction of drug ismore indicative of drug hypersensitivity than if the activation markeris not present. In order to obtain the information, sequencing is donebefore and after enrichment of cells by the particular marker. Thereforethe total as well as the fraction and number of clonotype cells with theparticular marker is measured.

An ADR is predicted without taking the drug. A high level of clonotypesthat interact with the drug or the presence of sequence motif likely tomount a strong response to the drug is detected to predict the ADRwithout administering the medication to the patient.

Similar immune response characteristics of drugs with idiosyncraticimmune-related ADRs are identified. These drugs may have high frequencyclonotypes that interact with the drugs or their metabolites. Drugs thatare likely to have immune-related ADRs are identified afteradministration in only a small number of patients.

The subclinical response of an ADR causing drug is detected by thedetermination of an increase in the clonotypes specific to drug (and/orits metabolite) after the administration of the medication.

Drugs that are likely to have an immune related ADR are identified bytracking the increase in the clonotypes corresponding to ARDs afteradministration in a small number of patients.

Example 28 Methods for Risk Stratification in Carotid Vascular Disease

Inflammation involved in the formation as and stability of the plaquesis detected in a patient. The immune response specific to vascularinflammation is used to indicate the risk of plaque destabilization.Specific antigens relevant in the immune reaction in the ICA (includingmodified or oxidized LDL and heat shock protein) are utilized toidentify the specific clonotypes relevant to immune reaction in ICA.Using similar procedures as described above T or B cell clonotypes thatinteract with the specific antigens are identified. The levels of theidentified, clonotypes are monitored to assess the risk of ICA plaquedestabilization.

Clonotypes that are relevant to ICA plaque destabilization are alsoidentified using an algorithm that is generated from a population studythat identifies characteristics that distinguish the relevant clonotypesfrom others. These relevant clonotypes are identified in the populationstudy by the virtue of their correlation with plaque destabilization orby their significant enrichment in the ICA plaque (for example asobtained from carotid endarterectomy) compared with blood. The sequencemotifs can be specific to distinct corresponding HLA genotypes. Thedeveloped algorithm are then be used to predict ICA plaquedestabilization relevant clonotypes in other patients.

Example 29 TCR Repertoire Analysis in EAE Mice

10 mice of SJL strain were treated with the peptide 139-151 along withcomplete Freund's adjuvant (CFA) using a commercially availableprotocol, e.g. Hooke Laboratories (Lawrence, Mass.). Eight of these micedeveloped Experimental Autoimmune Encephalitis (EAE), a mouse model ofmultiple sclerosis, and the other two did not. In addition 2 mice of thesame strain were treated with CFA only. For each mouse disease scoreswere obtained daily for 61 days after injection. The score range wasfrom 0 to 5. Blood samples were obtained before injection and specificdays afterwards. Overall, 11 blood samples were obtained from each mouseand the mice were sacrificed at day 62 or 63 and spleen, lymph nodes,and spinal cord was obtained. Blood and the tissue were immediatelystored in animal blood protect RNA reagent and RNAlater, respectively.RNA was extracted from the blood samples using Qiagen animal protectblood extraction kit and the tissue samples were mechanicallyhomogenized, and RNA prepared using RNA Qiagen Plus minikit cDNA wasproduced from each sample using Vilo cDNA synthesis kit (Lifetechnologies). The TCRβ repertoire from each of the samples wasamplified using the primers in Table 10.

TABLE 10  Mouse V Segment Primers* V Segment Primers SEQ ID NOCAAAGAGGTCAAATCTCTTCCCG 145 CTTATGGACAATCAGACTGCCTCA 146GTCATGGAGAAGTCTAAACTGTTTAAGG 147 GTAAACGAAACAGTTCCAAGGCG 148GGTGCCCAGTCGTTTTATACCTGAAT 149 CCCAGCAGATTCTCAGTCCAACAGT 150AGATATCCCTGATGGATACAAGGC 151 AGATATCCCTGATGGGTACAAGGC 152AGATGTCCCTGATGGGTACAAGGC 153 GATAATTCACAGTTGCCCTCGGAT 154GATGGTGGGGCTTTCAAGGATC 155 CAAGCTCCTATAGATGATTCAGGG 156CTATGATAAGATTTTGAACAGGGAAGC 157 GATCTACTATTCAATAACTGAAAACGATCTTC 158TAGCACTTTCTACTGTGAACTCAGCA 159 CTTGATCAAATAGACATGGTCAAGG 160AGAGATTCTCAGCTAAGTGTTCCTCG 161 GTTCTTCAGCAAATAGACATGACTG 162AGCGAAGGAGACATCCCTAAAGGAT 163 CGAGAGTGGATTCACCAAGGACAAG 164 *Each of theprimers have a common 14 bp (AGATCGGAAGAGCA) (SEQ ID NO 165) appended toits 5′ end.

A second PCR was performed on each of the samples using primers in Table7. Each sample was amplified with one pair of primers that amplified thecomplete set of first stage PCR amplicons but which also included anindividual sequence tag that could later allow individual samples to beidentified in a mixture of sequence data. A third PCR is done using onepair of primers for all samples (table 7). The purpose of the third PCRwas to ensure the integrity of the terminal sequences. The structure ofthe PCR product is shown in FIGS. 2A-2B and FIG. 3A. These PCR reactionswere pooled in sets of 64 such that each sample with the pool wasindexed by one of the unique tags incorporated in the second stage PCR.The molecules from these pools were then denatured and separated in twodimensions on a solid surface through hybridization with a flow cellcontaining oligonucleotides that hybridize with the terminal sequencesof the amplified products. The hybridized molecules were then amplifiedby a bridging amplification to form clusters on the two dimensionalsurface such that each cluster contained approximately 1,000 moleculeseach the result of an amplification of a single molecule from the PCRpool. A method was then used to cleave and release one of the twostrands of each of these molecules leaving a single stranded template. Asequencing primer was then hybridized to the clusters. Iterative roundsof sequencing were then carried out involving: the introduction of 4fluorescently labeled chemically terminated nucleotides of each nucleicacid base and a polymerase and buffers such that an incorporation of asingle nucleotide would occur for the active position of the extensionproduct that was complementary to each nucleotide; a washing step, afluorescence scan of the surface to measure which clusters incorporatedwhich fluorescent dye, the introduction of a cleavage chemical thatreleased the termination molecules from the incorporated bases alongwith the fluorescent label to allow for subsequent cycles; a wash step.These steps were iterated ˜100 times to reveal the sequence of eachcluster (read 1). The synthesized strand was then removed bydenaturation. A second primer was then introduced and the sequencingprocess re-iterated to read a 6 base tag. This synthesized strand wasthen removed by denaturation. The original template strand was thenallowed to hybridize back to the immobilized surface primers which wereextended to re-form the double stranded clusters. At this point theoriginal strand was cleaved resulting in clusters formed from thecomplements of the original single stranded clusters. A third sequencingprimer was introduced and it hybridized to this strand and thesequencing was iterated ˜60 cycles to obtain sequences from the reversestrand of the amplicons (read 2). The resulting sequences contributed100,000-200,000 reads per sample after having been sorted based on thetag sequences.

The obtained sequences were first mapped to specific regions of the Vand J segments. Specifically the first 27 bp of read 1 were mapped tothe last 27 bp of the different mouse J segments. Similarly the initialsequences of read2 were mapped to the sequences of the used primers.When a sequence is mapped to one of the primers then the sequence of theread2 following the primer is mapped to the different mouse V segments.Since Read1 is expected to reach the V segment, we also attempted to mapit in order to determine the frame and the amino acid sequence.Positions 81-95 of read 1 were mapped to the most 3′ 60 bp of thespecific V segment that read2 maps to. If a sequence does not havesubstantial identity to any of the J and V sequences it would beeliminated. Additionally the J and V mapping s are extended in read1.Since the specific J and V are mapped for each cluster we can assesswhether the bases outside the originally mapped sequence are consistentwith the sequence of the mapped V and J. These sequences will cease tobe consistent when the bases that result from the non-templatedreplication (N bases) or D segment are reached. Differences between readsequences and the V or J sequence in the originally mapped or theextended regions are then considered to be due to error and are“corrected”.

Reads that have identical sequences for positions 28-80 are thenconsidered a clonotype. Clonotypes that are very similar in sequence arethen assessed to whether they are likely to be independent clonotypes orone clonotypes that separated due to PCR and/or sequencing error. Wehave devised an algorithm that incorporates the frequency of the twoclonotypes under consideration the number of differences between themand the likelihood of error at that position to determine whether thetwo clonotypes are to be coalesced into one clonotype or not. When oneof the two clonotypes is at a very high frequency and the other is rareand there is only one base difference towards the end of the read (thatgenerally has more errors) then the two clonotypes are likely a resultof error and they are then coalesced. On the other hand the presence oftwo clonotypes at similar frequency and with three differences betweenthem indicates that these two clonotypes are genuinely independent andare not coalesced. The frequency of each TCR□ clonotype is thencomputed.

A public clonotype was present in all 12 mice. This clonotype had thesignature of a clonotype reactive to CFA. It was undetectable in any ofthe mice before the injection or at day 5, but it dramatically increasesto high frequency at later time points. Multiple nucleotide sequencesacross mice and within a single mouse coded for the same amino acidsequence of this clonotype. Overall and after eliminating clonotypesseen in less than 3/10 time points for a mouse (time point number 8 waseliminated from all the mice as there was unusual characteristics in afew of the mice), there are 23 observations of clonotypes with thisamino acid sequence (19 of which present at mean frequency >10-4) with10 distinct nucleotide sequences in the 12 mice (8 distinct sequencesfor the 19 high frequency clonotypes) were obtained (see table 10). Inaddition there are additional related clonotypes (with only one aminoacid difference) that also have the same pattern of being low beforeinjection and high after 10 days of the injection. This provides anoverwhelming evidence of a clonotype that is responsive to CFA.

TABLE 11  Mouse TCRβ public clonotype in response to CFA* Log10 cloneMouse mean SEQ SEQ ID frequency Nucleotide Sequence  ID NOProtein Sequence ID NO 62 −2.73861 CAAAATACAGCGTTTCTGCACTA 171FLCASSIAWGSAETLYF 193 CCCCAAGCTATACTGCTGGCACA GAGAAAA 62 −3.78089CAAAATACAGCGTTTCTGCACTC 172 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 34 −3.75307 CAAAATACAGCGTTTCTGCACTC 172FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 34 −3.2046CAAAATACAGCGTTTCTGCACTA 173 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 64 −2.95504 CAAAATACAGCGTTTCTGCACTC 174FLCASSIAWGSAETLYF 193 CCCCAAGCTATACTGCTGGCACA GAGAAAA 14 −3.10634CAAAATACAGCGTTTCTGCACTC 172 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 61 −3.07614 CAAAATACAGCGTTTCTGCACTA 171FLCASSIAWGSAETLYF 193 CCCCAAGCTATACTGCTGGCACA GAGAAAA 61 −4.62344CAAAATACAGCGTTTCTGCACTA 173 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 61 −4.6644 CAAAATACAGCGTTTCTGCACTT 175FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 55 −2.70719CAAAATACAGCGTTTCTGCACTC 172 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 55 −3.05281 CAAAATACAGCGTTTCTGCACTA 176FLCASSIAWGSAETLYF 193 CCCCAGGCAATACTGCTGGCAC AGAGAAAA 55 −3.44137CAAAATACAGCGTTTCTGCACTC 177 FLCASSIAWGSAETLYF 193 CCCCAGGCAATACTGCTGGCACAGAGAAAA 65 −3.73155 CAAAATACAGCGTTTCTGCACTG 178 FLCASSIAWGSAETLYF 193CCCCAAGCTATACTGCTGGCACA GAGAAAA 65 −3.42266 CAAAATACAGCGTTTCTGCACTA 173FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 35 −4.35749CAAAATACAGCGTTTCTGCACTG 179 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 35 −2.97796 CAAAATACAGCGTTTCTGCACTC 172FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 23 −3.20311CAAAATACAGCGTTTCTGCACTC 172 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 23 −4.70989 CAAAATACAGCGTTTCTGCACTA 173FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 11 −2.8685CAAAATACAGCGTTTCTGCACTC 172 FLCASSIAWGSAETLYF 193CCCCAGGCTATACTGCTGGCACA GAGAAAA 63 −3.38278 CAAAATACAGCGTTTCTGCACTA 173FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 63 −3.21617CAAAATACAGCGTTTCTGCACTC 174 FLCASSIAWGSAETLYF 193CCCCAAGCTATACTGCTGGCACA GAGAAAA 45 −3.79581 CAAAATACAGCGTTTCTGCACTC 172FLCASSIAWGSAETLYF 193 CCCCAGGCTATACTGCTGGCACA GAGAAAA 45 −3.86157CAAAATACAGCGTTTCTGCGCTC 180 FLCASSIAWGSAETLYF 193CCCCATGCTATACTGCTGGCACA GAGAAAA *The log10 mean frequency describes themean frequency among all 10 time points for the specific mouse.

While preferred embodiments of the present invention have been shown anddescribed herein, it is obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The public clonotype described in the above example was validated bylooking at TCR repertoires of additional mice. Specifically, thepresence of the clonotype in 11 PLP treated and 3 sham treated mice wereassessed. The clonotype was present in 12/14 mice. In addition therelated clonotype mentioned in the above example (with one amino aciddifference) was also seen repeatedly. There were 15 observations(different nucleotide sequences in the same animal or in differentanimals) for the clone with one amino acid difference. The clonotype wasnot seen in any of the animals before vaccination. The pattern offrequency was also quite similar to what was seen in the first set ofanimals. This validates that the identified public clonotype is indeedcommon to the majority of mice who were treated with the Freund'sadjuvant.

The use of affected tissue to help determine clonotypes relevant todisease. Clonotypes correlating with disease activity in the above EAEmodel were found. However, it is not readily obvious whether theclonotypes were specific to the adjuvant aspect of the vaccination orthe PLP peptide. The presence or absence of the clonotypes in the shamtreated samples was a method to distinguish the two possibilities. Aswas disclosed previously, affected tissue can be used to determineclonotypes relevant to the disease. In this case, spinal cord tissue wasavailable. Clonotypes were filtered from the data which had a frequencythat is at least 3 larger in spinal cord than each of the three othersamples obtained at the same time (spleen, lymph node, and blood).Clonotypes were then examined that were correlated with the diseasescore. A clonotype was identified that appeared more than once. Thesequence of the clonotype was LYCTCSALGGSSYEQYF (sequence A) (SEQ ID NO:194). This sequence was looked for and identified it in all 8 mice withthe disease, Two mice that were treated with PLP but did not developdisease and it was not detected in the two sham-treated mice. Thepattern of frequency of the clonotype in the serial blood samplesdiffered between mice. However, in all mice the clonotype was absentbefore vaccination. In addition the pattern of higher frequency in thespinal cord than the other tissue at the same time point wasdemonstrated in all except one mouse.

Given the risk of overfitting, an independent set of mice was examinedto validate these finding. The presence of sequence A in additional micewas examined. Specifically, the presence of sequence A was tested in thefollowing circumstances: in eight additional PLP treated mice thatdeveloped EAE, three PLP treated mice that did not develop EAE, andthree sham treated mice. Among the 11 PLP-treated mice, the clonotypewas present in eight mice. Two out of the three that did not have theclonotype did not develop disease. The clonotype was not found in thethree sham treated mice nor was it found in any of the PLP-treated miceat the point before vaccination. In addition, the pattern of higherfrequency in the spinal cord than the other tissues of the same timepoint was demonstrated in all the animals. In all the samples (theinitial discovery and later validation) among 21 animals treated withPLP, 18 have the clones, but none before vaccination. In addition noneof the five sham treated mice have sequence A. This provides strongevidence for a public EAE clonotype that is specific to the disease.

Additional clonotypes that correlate with disease and are enriched inthe spinal cord appear to have very similar sequences to each other.These semi-private clonotypes are additional disease-specificclonotypes. These can be more comprehensively identified through a theutilization of a motif finding algorithm to determine shared sequencesamong clonotypes enriched in spinal cord and correlate with disease.

Example 30 Somatic Hypermutations Identified in IgH Clonotype Profiling

Three sets of amplifications were performed using IgH V segment primerslisted in table 5 and the primers complementary to the IgG constantsequence that are also disclosed above. cDNA from 7 normal samples, 7samples from patients with multiple sclerosis, and 4 samples with SLEwere used as templates for amplification. After second stage PCR thatintroduced a unique tag for each sample, the products were spatiallyisolated and subjected to sequencing.

The sequences were then mapped to individual V and J segments andassembled into clonotypes using methodologies disclosed above. We soughtevidence of frequent highly related clonotypes that may be the result ofsomatic hypermutation in these samples. Data in table 11 shows astriking example in one of the multiple sclerosis patients. In thisexample, 12 distinct clonotypes were identified. These 12 nucleotideclonotypes code for 3 highly related amino acid sequences. Two of theamino acid sequences are at very high frequency (>1%) and differ by oneconserved amino acid (Lysine vs. Arginine).

TABLE 12  List of related clonotypes in a multiple sclerosis patient*Frequency SEQ SEQ ( ) Consensus Sequence ID NO Protein Sequence ID NO0.095834689 CTGGCCCCAATTCCATCTGCCTG 181 TAVYYCTCFTGRWNWGQ 195TAAAGCATGTACAGTAATACACA GCCGTGT 2.098569825 CTGGCCCCAGTTCCATTTGGTAG 182TAVYYCTCFTTKWNWGQ 196 TAAAACATGTACAGTAATACACA GCCGTGT 0.856216488CTGGCCCCAATTCCACTTGGTAG 183 TAVYYCTCFTTKWNWGQ 196TAAAACATGTACAGTAATACACA GCCGTGT 0.785914399 CTGGCCCCAATTCCACTTGGTGG 184TAVYYCTCFTTKWNWGQ 196 TAAAACATGTACAATAATAGACA GCCGTGT 0.395930174CTGGCCCCAGTTCCATTTGGTAG 185 TAVYYCTCFTTKWNWGQ 196TAAAACATGTACAGTAATATACA GCAGTGT 0.128712283 CTGGCCCCAGTTCCATTTGGTAG 186TAVYYCTCFTTKWNWGQ 196 TAAAACATGTACAGTAATATACA GCTGTGT 0.094435642CTGGCCCCAGTTCCATTTGGTAG 187 TAVYYCTCFTTKWNWGQ 196TAAAACATGTACAGTAATATACA GCGGTGT 0.053513531 CTGGCCCCAATTCCACTTGGTAG 188TAVYYCTCFTTKWNWGQ 196 TAAAACATGTACAGTAATAGAC AGCAGTGT 0.019936413CTGGCCCCAGTTCCATTTGGTAG 189 TAVYYCTCFTTKWNWGQ 196TAAAACATGTACAGTAATATACG GCCGTGT 0.014340227 CTGGCCCCAGTTCCATTTGGTAG 190TAVYYCTCFTTKWNWGQ 196 TAAAACATGTACAGTAATATACA GCCGTTG 1.305310431CTGGCCCCAGTTCCATCTCGTAG 191 TAVYYCTCFTTRWNWGQ 197TAAAACATGTACAATAATACACA GCCGTGT 0.199364133 CTGGCCCCAGTTCCATCTGGTAG 192TAVYYCTCFTTRWNWGQ 197 TAAAACACGTACAATAATACACA GCCGTGT *The sequence ofpositions 28-80 is shown with the amino acid sequence. The frequencycolumn notes the frequency of the clonotype in the sample. For examplethe second clonotypes represent over 2% of all the sequences in thissample.

Example 31 Forensic Use of Immune Profiling

Clonotype profiles for T and/or B cell receptors may be employed forhuman and animal identification. The tremendous diversity of these ofclonotype profiles provides a very unique signature of the individual.This is exemplified by profiles of FIG. 10. In this example TCRβsequences were amplified by reverse transcriptase PCR from mRNAextracted from the blood of two different individuals. The primerscomplementary to the V regions are listed in Table 13. The second stageamplification primers are the same as those of Example 2. The productswere sequenced and the frequency of each clonotype frequency determined.As can be seen in FIG. 10, the vast majority of clonotypes from theindividuals are different, clonotypes of one individual (sample 122)positioned almost exclusively along the X axis and clonotypes of theother individual (sample 140) positioned almost exclusively along the Yaxis. Only about 25 clonotypes appear to be shared, as indicated byoff-axis data points. This disparity of clonotype usage betweenindividuals has been corroborated by Warren et al, Genome Research, Epub(24 Feb. 2011). In one aspect of the invention, this disparity inclonotype usage provides a method for distinguishing individualscomprising the steps: (a) obtaining a clonotype profile of a firstsample, (b) obtaining a clonotype profile of a second sample, and (c)determining whether the first sample and the second sample are from thesame individual by measuring the degree to which usage of clonotypesoverlaps.

TABLE 13  Human TCRβ V segment primers V Segment Primer SEQ ID NOAACTATGTTTTGGTATCGTCAGT 2 TTCTGGTACCGTCAGCAAC 3 AGTGTATCCTGGTACCAACAG 4AGTGTGTACTGGTACCAACAG 5 ACTGTGTCCTGGTACCAACAG 6 AGTGTGTCCTGGTACCAACAG 7TCTGTGTACTGGTACCAACAG 8 CCCTTTACTGGTACCGACAG 9 GCCTTTATTGGTACCGACAG 166CCCTTTACTGGTACCGACAAA 11 CCCTTTATTGGTACCGACAG 167 TTTTGGTACCAACAGGTCC 12TTTTGGTACCAACAGGCCC 13 AACCCTTTATTGGTATCAACAG 14 CGCTATGTATTGGTACAAGCA15 GGCAATGTATTGGTACAAGCA 16 GGCTATGTATTGGTACAAGCA 168TTTCTGGTACAGACAGACCATGA 17 TACTATGTATTGGTATAAACAGGACTC 18CAAAATGTACTGGTATCAACAA 19 ATGTTCTGGTATCGACAAGACC 20ATGTACTGGTATCGACAAGACC 21 TGCCATGTACTGGTATAGACAAG 22GTATCGACAAGACCCAGGCA 169 ATGTCCTGGTATCGACAAGACC 170TAATCTTTATTGGTATCGACGTGT 27 GCCATGTACTGGTACCGACA 28TCATGTTTACTGGTATCGGCAG 29 CAACCTATACTGGTACCGACA 30CATGCTACCCTTTACTGGTACC 31 CACAATACCCTTTACTGGTACC 32ATACTTCTATTGGTACAGACAAATCT 33 CACTGTCTACTGGTACCAGCA 34CGTCATGTACTGGTACCAGCA 35 * All the primers have a common 14 bp(AGATCGGAAGAGCA) (SEQ ID NO 165) appended to their 5′ end.Of 342 clonotypes in blood from the first sample (sample 122) at afrequency >10⁻⁴ only one is detected in the second individual (sample140). Reciprocally, out of 505 clonotypes present at frequency >10-4 inthe sample 144, 3 are detected in the sample 122. As a control forrandom fluctuation of the measurement, out of the same 505 clonotypes,504 clonotypes were present in a replicate amplification sample. Thisdemonstrates the potential for a complete clonotype profile of being apotentially extremely specific identifier. Of course, the nature ofthese profiles is that they are not stable in time as new immunereactions will add new clonotypes to the spectrum. These processeshowever do not alter these profiles very rapidly. It can be seen thatwhile the precise frequency of individual clonotypes shift in time, theset of clonotypes present at a measurable frequency is likely muchgreater than what would be found in a second individual. Algorithms canbe developed to define the fraction and number of identical clonotypesthat would be necessary to determine that two specimens are from thesame individual. Furthermore, because this diversity is found in a genewith a great deal of active function in contrast to microsatellitediversity, it is possible to extract potentially relevant identificationinformation without needing to have a matched sample from an identifieddonor sample. Information about the health of this individual, his orher vaccination history, etc. is measurable from the clonotypeinformation.

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. The present invention is applicableto a variety of sensor implementations and other subject matter, inaddition to those discussed above.

DEFINITIONS

Unless otherwise specifically defined herein, terms and symbols ofnucleic acid chemistry, biochemistry, genetics, and molecular biologyused herein follow those of standard treatises and texts in the field,e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman,New York, 1992); Lehninger, Biochemistry, Second Edition (WorthPublishers, New York, 1975); Strachan and Read, Human MolecularGenetics, Second Edition (Wiley-Liss, New York, 1999); Abbas et al,Cellular and Molecular Immunology, 6^(th) edition (Saunders, 2007).

“Amplicon” means the product of a polynucleotide amplification reaction;that is, a clonal population of polynucleotides, which may be singlestranded or double stranded, which are replicated from one or morestarting sequences. The one or more starting sequences may be one ormore copies of the same sequence, or they may be a mixture of differentsequences. Preferably, amplicons are formed by the amplification of asingle starting sequence. Amplicons may be produced by a variety ofamplification reactions whose products comprise replicates of the one ormore starting, or target, nucleic acids. In one aspect, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al,U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491 “NASBA”);Lizardi, U.S. Pat. No. 5,854,033; Aono et al, Japanese patent publ. JP4-262799 (rolling circle amplification); and the like. In one aspect,amplicons of the invention are produced by PCRs. An amplificationreaction may be a “real-time” amplification if a detection chemistry isavailable that permits a reaction product to be measured as theamplification reaction progresses, e.g. “real-time PCR” described below,or “real-time NASBA” as described in Leone et al, Nucleic AcidsResearch, 26: 2150-2155 (1998), and like references. As used herein, theterm “amplifying” means performing an amplification reaction. A“reaction mixture” means a solution containing all the necessaryreactants for performing a reaction, which may include, but not belimited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like.

“Clonality” as used herein means a measure of the degree to which thedistribution of clonotype abundances among clonotypes of a repertoire isskewed to a single or a few clonotypes. Roughly, clonality is an inversemeasure of clonotype diversity. Many measures or statistics areavailable from ecology describing species-abundance relationships thatmay be used for clonality measures in accordance with the invention,e.g. Chapters 17 & 18, in Pielou, An Introduction to MathematicalEcology, (Wiley-Interscience, 1969). In one aspect, a clonality measureused with the invention is a function of a clonotype profile (that is,the number of distinct clonotypes detected and their abundances), sothat after a clonotype profile is measured, clonality may be computedfrom it to give a single number. One clonality measure is Simpson'smeasure, which is simply the probability that two randomly drawnclonotypes will be the same. Other clonality measures includeinformation-based measures and McIntosh's diversity index, disclosed inPielou cited above).

“Clonotype” means a recombined nucleotide sequence of a T cell or B cellencoding a T cell receptor (TCR) or B cell receptor (BCR), or a portionthereof. In one aspect, a collection of all the distinct clonotypes of apopulation of lymphocytes of an individual is a repertoire of suchpopulation, e.g. Arstila et al, Science, 286: 958-961 (1999); Yassai etal, Immunogenetics, 61: 493-502 (2009); Kedzierska et al, Mol. Immunol.,45(3): 607-618 (2008); and the like. As used herein, “clonotypeprofile,” or “repertoire profile,” is a tabulation of clonotypes of asample of T cells and/or B cells (such as a peripheral blood samplecontaining such cells) that includes substantially all of therepertoire's clonotypes and their relative abundances. “Clonotypeprofile,” “repertoire profile,” and “repertoire” are used hereininterchangeably. That is, the term “repertoire,” as discussed more fullybelow, means a repertoire measured from a sample of lymphocytes). In oneaspect of the invention, clonotypes comprise portions of animmunoglobulin heavy chain IgH) or a TCR β chain. In other aspects ofthe invention, clonotypes may be based on other recombined molecules,such as immunoglobulin light chains or TCRα chains, or portions thereof.

Complementarity determining regions” (CDRs) mean regions of animmunoglobulin (i.e., antibody) or T cell receptor where the moleculecomplements an antigen's conformation, thereby determining themolecule's specificity and contact with a specific antigen. T cellreceptors and immunoglobulins each have three CDRs: CDR1 and CDR2 arefound in the variable (V) domain, and CDR3 includes some of V, all ofdiverse (D) (heavy chains only) and joint (J), and some of the constant(C) domains.

“Internal standard” means a nucleic acid sequence that is amplified inthe same amplification reaction as one or more target polynucleotides inorder to permit absolute or relative quantification of the targetpolynucleotides in a sample. An internal standard may be endogenous orexogenous. That is, an internal standard may occur naturally in thesample, or it may be added to the sample prior to amplification. In oneaspect, multiple exogenous internal standard sequences may be added to areaction mixture in a series of predetermined concentrations to providea calibration to which a target amplicon may be compared to determinethe quantity of its corresponding target polynucleotide in a sample.Selection of the number, sequences, lengths, and other characteristicsof exogenous internal standards is a routine design choice for one ofordinary skill in the art. Preferably, endogenous internal standards,also referred to herein as “reference sequences,” are sequences naturalto a sample that correspond to minimally regulated genes that exhibit aconstant and cell cycle-independent level of transcription, e.g. Selveyet al, Mol. Cell Probes, 15: 307-311 (2001). Exemplary referencesequences include, but are not limited to, sequences from the followinggenes: GAPDH, β₂-microglobulin, 18S ribosomal RNA, and β-actin (althoughsee Selvey et al, cited above).

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., primers,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains primers.

“Lymphoid neoplasm” means an abnormal proliferation of lymphocytes thatmay be malignant or non-malignant. A lymphoid cancer is a malignantlymphoid neoplasm. Lymphoid neoplasms are the result of, or areassociated with, lymphoproliferative disorders, including but notlimited to, follicular lymphoma, chronic lymphocytic leukemia (CLL),acute lymphocytic leukemia (ALL), hairy cell leukemia, lymphomas,multiple myeloma, post-transplant lymphoproliferative disorder, mantlecell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), T celllymphoma, or the like, e.g. Jaffe et al, Blood, 112: 4384-4399 (2008);Swerdlow et al, WHO Classification of Tumours of Haematopoietic andLymphoid Tissues (e. 4^(th)) (IARC Press, 2008).

“Minimal residual disease” means remaining cancer cells after treatment.The term is most frequently used in connection with treatment oflymphomas and leukemias.

“Pecent homologous,” “percent identical,” or like terms used inreference to the comparison of a reference sequence and another sequence“comparison sequence”) mean that in an optimal alignment between the twosequences, the comparison sequence is identical to the referencesequence in a number of subunit positions equivalent to the indicatedpercentage, the subunits being nucleotides for polynucleotidecomparisons or amino acids for polypeptide comparisons. As used herein,an “optimal alignment” of sequences being compared is one that maximizesmatches between subunits and minimizes the number of gaps employed inconstructing an alignment. Percent identities may be determined withcommercially available implementations of algorithms, such as thatdescribed by Needleman and Wunsch, J. Mol. Biol., 48: 443-453 1970)“GAP” program of Wisconsin Sequence Analysis Package, Genetics ComputerGroup, Madison, Wis.), or the like. Other software packages in the artfor constructing alignments and calculating percentage identity or othermeasures of similarity include the “BestFit” program, based on thealgorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981) (Wisconsin Sequence Analysis Package, Genetics ComputerGroup, Madison, Wis.). In other words, for example, to obtain apolynucleotide having a nucleotide sequence at least 95 percentidentical to a reference nucleotide sequence, up to five percent of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to five percent of thetotal number of nucleotides in the reference sequence may be insertedinto the reference sequence.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C. The term “PCR” encompasses derivativeforms of the reaction, including but not limited to, RT-PCR, real-timePCR, nested PCR, quantitative PCR, multiplexed PCR, and the like.Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to afew hundred μL, e.g. 200 μL. “Reverse transcription PCR,” or “RT-PCR,”means a PCR that is preceded by a reverse transcription reaction thatconverts a target RNA to a complementary single stranded DNA, which isthen amplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038, which patentis incorporated herein by reference. “Real-time PCR” means a PCR forwhich the amount of reaction product, i.e. amplicon, is monitored as thereaction proceeds. There are many forms of real-time PCR that differmainly in the detection chemistries used for monitoring the reactionproduct, e.g. Gelfand et al, U.S. Pat. No. 5,210,015 “taqman”); Wittweret al, U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes);Tyagi et al, U.S. Pat. No. 5,925,517 (molecular beacons); which patentsare incorporated herein by reference. Detection chemistries forreal-time PCR are reviewed in Mackay et al, Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228(1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. Typically, the number oftarget sequences in a multiplex PCR is in the range of from 2 to 50, orfrom 2 to 40, or from 2 to 30. “Quantitative PCR” means a PCR designedto measure the abundance of one or more specific target sequences in asample or specimen. Quantitative PCR includes both absolute quantitationand relative quantitation of such target sequences. Quantitativemeasurements are made using one or more reference sequences or internalstandards that may be assayed separately or together with a targetsequence. The reference sequence may be endogenous or exogenous to asample or specimen, and in the latter case, may comprise one or morecompetitor templates. Typical endogenous reference sequences includesegments of transcripts of the following genes: β-actin, GAPDH,β₂-microglobulin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al, Biotechniques, 26: 112-126 (1999);Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al,Gene, 122: 3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research,17: 9437-9446 (1989); and the like.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2^(nd) Edition (Cold SpringHarbor Press, New York, 2003).

“Quality score” means a measure of the probability that a baseassignment at a particular sequence location is correct. A varietymethods are well known to those of ordinary skill for calculatingquality scores for particular circumstances, such as, for bases calledas a result of different sequencing chemistries, detection systems,base-calling algorithms, and so on. Generally, quality score values aremonotonically related to probabilities of correct base calling. Forexample, a quality score, or Q, of 10 may mean that there is a 90percent chance that a base is called correctly, a Q of 20 may mean thatthere is a 99 percent chance that a base is called correctly, and so on.For some sequencing platforms, particularly those usingsequencing-by-synthesis chemistries, average quality scores decrease asa function of sequence read length, so that quality scores at thebeginning of a sequence read are higher than those at the end of asequence read, such declines being due to phenomena such as incompleteextensions, carry forward extensions, loss of template, loss ofpolymerase, capping failures, deprotection failures, and the like.

“Repertoire”, or “immune repertoire”, means a set of distinct recombinednucleotide sequences that encode T cell receptors (TCRs) or B cellreceptors (BCRs), or fragments thereof, respectively, in a population oflymphocytes of an individual, wherein the nucleotide sequences of theset have a one-to-one correspondence with distinct lymphocytes or theirclonal subpopulations for substantially all of the lymphocytes of thepopulation. In one aspect, a population of lymphocytes from which arepertoire is determined is taken from one or more tissue samples, suchas one or more blood samples. A member nucleotide sequence of arepertoire is referred to herein as a “clonotype.” In one aspect,clonotypes of a repertoire comprises any segment of nucleic acid commonto a T cell or a B cell population which has undergone somaticrecombination during the development of TCRs or BCRs, including normalor aberrant (e.g. associated with cancers) precursor molecules thereof,including, but not limited to, any of the following: an immunoglobulinheavy chain (IgH) or subsets thereof (e.g. an IgH variable region, CDR3region, or the like), incomplete IgH molecules, an immunoglobulin lightchain or subsets thereof (e.g. a variable region, CDR region, or thelike), T cell receptor α chain or subsets thereof, T cell receptor βchain or subsets thereof (e.g. variable region, CDR3, V(D)J region, orthe like), a CDR (including CDR1, CDR2 or CDR3, of either TCRs or BCRs,or combinations of such CDRs), V(D)J regions of either TCRs or BCRs,hypermutated regions of IgH variable regions, or the like. In oneaspect, nucleic acid segments defining clonotypes of a repertoire areselected so that their diversity (i.e. the number of distinct nucleicacid sequences in the set) is large enough so that substantially every Tcell or B cell or clone thereof in an individual carries a uniquenucleic acid sequence of such repertoire. That is, in accordance withthe invention, a practitioner may select for defining clonotypes aparticular segment or region of recombined nucleic acids that encodeTCRs or BCRs that do not reflect the full diversity of a population of Tcells or B cells; however, preferably, clonotypes are defined so thatthey do reflect the diversity of the population of T cells and/or Bcells from which they are derived. That is, preferably each differentclone of a sample has different clonotype. (Of course, in someapplications, there will be multiple copies of one or more particularclonotypes within a profile, such as in the case of samples fromleukemia or lymphoma patients). In other aspects of the invention, thepopulation of lymphocytes corresponding to a repertoire may becirculating B cells, or may be circulating T cells, or may besubpopulations of either of the foregoing populations, including but notlimited to, CD4+ T cells, or CD8+ T cells, or other subpopulationsdefined by cell surface markers, or the like. Such subpopulations may beacquired by taking samples from particular tissues, e.g. bone marrow, orlymph nodes, or the like, or by sorting or enriching cells from a sample(such as peripheral blood) based on one or more cell surface markers,size, morphology, or the like. In still other aspects, the population oflymphocytes corresponding to a repertoire may be derived from diseasetissues, such as a tumor tissue, an infected tissue, or the like. In oneembodiment, a repertoire comprising human TCR β chains or fragmentsthereof comprises a number of distinct nucleotide sequences in the rangeof from 0.1×10⁶ to 1.8×10⁶, or in the range of from 0.5×10⁶ to 1.5×10⁶,or in the range of from 0.8×10⁶ to 1.2×10⁶. In another embodiment, arepertoire comprising human IgH chains or fragments thereof comprises anumber of distinct nucleotide sequences in the range of from 0.1×10⁶ to1.8×10⁶, or in the range of from 0.5×10⁶ to 1.5×10⁶, or in the range offrom 0.8×10⁶ to 1.2×10⁶. In a particular embodiment, a repertoire of theinvention comprises a set of nucleotide sequences encoding substantiallyall segments of the V(D)J region of an IgH chain. In one aspect,“substantially all” as used herein means every segment having a relativeabundance of 0.001 percent or higher; or in another aspect,“substantially all” as used herein means every segment having a relativeabundance of 0.0001 percent or higher. In another particular embodiment,a repertoire of the invention comprises a set of nucleotide sequencesthat encodes substantially all segments of the V D)J region of a TCR βchain. In another embodiment, a repertoire of the invention comprises aset of nucleotide sequences having lengths in the range of from 25-200nucleotides and including segments of the V, D, and J regions of a TCR βchain. In another embodiment, a repertoire of the invention comprises aset of nucleotide sequences having lengths in the range of from 25-200nucleotides and including segments of the V, D, and J regions of an IgHchain. In another embodiment, a repertoire of the invention comprises anumber of distinct nucleotide sequences that is substantially equivalentto the number of lymphocytes expressing a distinct IgH chain. In anotherembodiment, a repertoire of the invention comprises a number of distinctnucleotide sequences that is substantially equivalent to the number oflymphocytes expressing a distinct TCR β chain. In still anotherembodiment, “substantially equivalent” means that with ninety-ninepercent probability a repertoire of nucleotide sequences will include anucleotide sequence encoding an IgH or TCR β or portion thereof carriedor expressed by every lymphocyte of a population of an individual at afrequency of 0.001 percent or greater. In still another embodiment,“substantially equivalent” means that with ninety-nine percentprobability a repertoire of nucleotide sequences will include anucleotide sequence encoding an IgH or TCR β or portion thereof carriedor expressed by every lymphocyte present at a frequency of 0.0001percent or greater. The sets of clonotypes described in the foregoingtwo sentences are sometimes referred to herein as representing the “fullrepertoire” of IgH and/or TCRβ sequences. As mentioned above, whenmeasuring or generating a clonotype profile (or repertoire profile), asufficiently large sample of lymphocytes is obtained so that suchprofile provides a reasonably accurate representation of a repertoirefor a particular application. In one aspect, samples comprising from 10⁵to 10′ lymphocytes are employed, especially when obtained fromperipheral blood samples of from 1-10 mL.

“Sequence tag” or “tag”) means an oligonucleotide that is attached to apolynucleotide or template and is used to identify and/or track thepolynucleotide or template in a reaction. An oligonucleotide tag may beattached to the 3′- or 5′-end of a polynucleotide or template or it maybe inserted into the interior of such polynucleotide template to form alinear conjugate, sometime referred to herein as a “taggedpolynucleotide,” or “tagged template,” or “tag-polynucleotideconjugate,” or the like. Oligonucleotide tags may vary widely in sizeand compositions; the following references provide guidance forselecting sets of oligonucleotide tags appropriate for particularembodiments: Brenner, U.S. Pat. No. 5,635,400; Brenner et al, Proc.Natl. Acad. Sci., 97: 1665-1670 (2000); Church et al, European patentpublication 0 303 459; Shoemaker et al, Nature Genetics, 14: 450-456(1996); Morris et al, European patent publication 0799897A1; Wallace,U.S. Pat. No. 5,981,179; and the like. Lengths and compositions ofoligonucleotide tags can vary widely, and the selection of particularlengths and/or compositions depends on several factors including,without limitation, how tags are used to generate a readout, e.g. via ahybridization reaction or via an enzymatic reaction, such as sequencing;whether they are labeled, e.g. with a fluorescent dye or the like; thenumber of distinguishable oligonucleotide tags required to unambiguouslyidentify a set of polynucleotides, and the like, and how different musttags of a set be in order to ensure reliable identification, e.g.freedom from cross hybridization or misidentification from sequencingerrors. In one aspect, oligonucleotide tags can each have a lengthwithin a range of from 2 to 36 nucleotides, or from 4 to 30 nucleotides,or from 8 to 20 nucleotides, or from 6 to 10 nucleotides, respectively.In one aspect, sets of tags are used wherein each oligonucleotide tag ofa set has a unique nucleotide sequence that differs from that of everyother tag of the same set by at least two bases; in another aspect, setsof tags are used wherein the sequence of each tag of a set differs fromthat of every other tag of the same set by at least three bases.

1-61. (canceled)
 62. A method of monitoring a lymphoid neoplasm in apatient with defined cancer clonotypes comprising the steps of: (a)determining a clonotype profile from recombined DNA sequences in T-cellsand/or B-cells comprising the steps of: (i) obtaining from the patient asample comprising T-cells and/or B-cells; (ii) amplifying molecules ofnucleic acid from the T-cells and/or B-cells of the sample; (iii)spatially isolating individual molecules of the amplified nucleic acid;(iv) sequencing the individual molecules of nucleic acid to providesequence reads and forming clonotypes therefrom, and (b) evaluating theclonotype profile for levels of the cancer clonotypes defined for thepatient and clonotypes evolved therefrom.
 63. The method of claim 62wherein said evolved clonotypes are related to said cancer clonotypes bysomatic rearrangement.
 64. The method of claim 63 wherein said somaticrearrangement is a VH replacement or an NDN replacement whereby saidevolved clonotype has identically mutated V region and J region as saidcancer clonotype but having a different NDN region as said cancerclonotype.
 65. The method of claim 64 further including the steps of:repeating said steps (a) and (b) with a successive sample from saidpatient; evaluating a clonotype profile of the successive sample for apresence, absence and/or level of said cancer clonotypes and clonotypesevolved therefrom; and determining a disease status of said lymphoidneoplasm from an increase or decrease of frequencies of said cancerclonotypes and clonotypes evolved therefrom.
 66. The method of 65wherein said sample and said successive samples are obtained fromperipheral blood or bone marrow of said patient and wherein said step ofsequencing provides at least 10⁴ sequence reads.
 67. The method of claim64 wherein in said step of sequencing said sequence reads are coalescedinto different clonotypes whenever said sequence reads are distinct witha confidence of at least 99.9 percent.
 68. The method of claim 67wherein said step of sequencing provides at least 10¹ sequence reads.69. The method of claim 64 wherein in said step of sequencing at least aplurality of said sequence reads are combined to form each clonotype.70. The method of claim 69 wherein said plurality is 10 or more andwherein said step of sequencing provides at least 10⁴ sequence reads.71. The method of claim 64 wherein said clonotype profile comprisesevery clonotype of said sample present at a frequency of 0.01 percent orgreater with a probability of ninety-nine percent.
 72. The method ofclaim 71 wherein in said step of sequencing at least a plurality of saidsequence reads are combined to form each clonotype and said step ofsequencing provides at least 10⁴ sequence reads each having a length ofat least 30 bp.
 73. The method of claim 62 wherein said evolvedclonotypes are at least ninety percent identical to said cancerclonotypes.
 74. The method of claim 73 wherein said clonotypes areformed from said sequence reads having lengths in the range of from 20to 400 nucleotides.
 75. The method of claim 62 wherein said evolvedclonotypes are related to said cancer clonotypes by somatichypermutation.
 76. The method of claim 62 wherein said clonotypescomprise a rearrangement or a portion of a rearrangement selected fromthe group consisting of a VDJ rearrangement of NH, a DJ rearrangement ofIgH, a VJ rearrangement of IgK, a VJ rearrangement of IgL, a VDJrearrangement of ICR β, a DJ rearrangement of TCR β, a VJ rearrangementof TCR α, a VJ rearrangement of TCRγ, a VDJ rearrangement of TCR δ, anda VD rearrangement of TCR δ.
 77. A method a of monitoring a lymphoidneoplasm in a patient with defined cancer clonotypes comprising thesteps of: (a) obtaining from the patient a sample comprising T-cellsand/or B-cells; (b) amplifying molecules of recombined nucleic acid fromthe T-cells and/or B-cells of the sample; (c) spatially isolatingindividual a molecules of the amplified recombined nucleic acid; (d)sequencing the individual molecules of recombined nucleic acid toprovide sequence reads and forming clonotypes therefrom; and (e)evaluating the clonotypes for levels of the cancer clonotypes definedfor the patient and clonotypes evolved therefrom.
 78. The method ofclaim 77 further including the steps of: repeating said steps (a) and(b) with a successive sample from said patient; evaluating clonotypes ofthe successive sample for a presence, absence and/or level of saidcancer clonotypes and clonotypes evolved therefrom; and determining adisease status of said lymphoid neoplasm from an increase or decrease offrequencies of said cancer clonotypes and clonotypes evolved therefrom.79. The method of 78 wherein said sample and said successive sample areobtained from peripheral blood or bone marrow of said patient andwherein said step of sequencing provides at least 10⁴ sequence reads.80. The method of claim 77 wherein said evolved clonotypes are relatedto said cancer clonotypes by somatic rearrangement.
 81. The method ofclaim 80 wherein said somatic rearrangement is a VH replacement or anNDN replacement whereby said evolved clonotype has identically mutated Vregion and J region as said cancer clonotype but having a different NDNregion as said cancer clonotype.
 82. The method of claim 81 wherein insaid step of sequencing said sequence: reads arc coalesced intodifferent clonotypes whenever said sequence reads are distinct with aconfidence of at least 99.9 percent.
 83. The method of claim 82 whereinsaid step of sequencing provides at least 10⁴ sequence reads.
 84. Themethod of claim 81 wherein in said step of sequencing at least aplurality of said sequence reads are combined to form each clonotype.85. The method of claim 84 wherein said plurality is 10 or more andwherein said step of sequencing provides at least 10⁴ sequence reads.86. A method of monitoring a lymphoid neoplasm in a patient with definedcancer clonotypes comprising the steps of: (a) obtaining from thepatient a sample comprising T-cells and/or B-cells; (b) amplifyingmolecules of recombined nucleic acid from the 1-cells and/or B-cells ofthe sample; (c) spatially isolating individual molecules of theamplified recombined nucleic acid; (d) sequencing the individualmolecules of recombined nucleic acid to provide at least 10⁴ sequencereads and forming clonotypes therefrom by combining at least a pluralityof sequence reads for each clonotype; and (e) evaluating the clonotypesfor levels of the cancer clonotypes defined for the patient andclonotypes evolved therefrom.
 87. The method of 86 wherein in said stepof sequencing said sequence reads are combined into different clonotypeswhenever said sequence reads are distinct with a confidence of at least99.9 percent.
 88. The method of claim 87 wherein said clonotypescomprise a rearrangement or a portion of a rearrangement selected fromthe group consisting of a VDJ rearrangement of IgH, a DJ rearrangementof IgH, a VJ rearrangement of IgK, a VJ rearrangement of IgL, a VDJrearrangement of TCR β, a DJ rearrangement of TCR β, a VJ rearrangementof TCR α, a VJ rearrangement, of ICRγ, a VDJ rearrangement of TCR δ, anda VD rearrangement of TCR δ.
 89. The method of claim 87 wherein saidcancer clonotypes include a cross lineage rearrangement.
 90. The methodof claim 87 wherein said cancer clonotypes include an inactive immunereceptor.